Oligonucleotide compounds for treatment of preeclampsia and other angiogenic disorders

ABSTRACT

This disclosure relates to novel targets for angiogenic disorders. Novel oligonucleotides are also provided. Methods of using the novel oligonucleotides for the treatment of angiogenic disorders (e.g., preeclampsia) are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/814,350, filed Nov. 15, 2017, which is a continuation of U.S. patentapplication Ser. No. 15/089,437, filed Apr. 1, 2016, now U.S. Pat. No.9,862,952, which claims priority to U.S. Provisional Patent ApplicationSer. Nos. 62/291,961, filed Feb. 5, 2016; 62/291,678, filed Feb. 5,2016; and 62/142,745, filed Apr. 3, 2015. The entire contents of theseapplications are herein incorporated by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant No.OPP1086170 awarded by the National Science Foundation. The Governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 5, 2019, isnamed 618244_UM9-208CON2_Sequence_Listing.txt and is 150,763 bytes insize.

FIELD OF THE INVENTION

This disclosure relates to novel angiogenic targets and noveloligonucleotide compounds for the treatment of angiogenic disorders(e.g., preeclampsia).

BACKGROUND

Complicating 5-8% of all pregnancies, preeclampsia (PE) is one of thethree main causes of premature birth. The most notable characteristicsof PE are hypertension, edema and excess protein in the urine(proteinuria) after the 20th week of pregnancy. Consequences for thefetus can be grave, ranging from small-for-gestational-age infancy tohypoxia-induced neurologic injury (e.g., cerebral palsy) to death.Maternal complications include renal failure, HELLP syndrome (Hemolysis,Elevated Liver enzymes, and Low Platelets), seizures, stroke, and death.PE and related hypertensive disorders are conservatively estimated tocause 76,000 maternal and 500,000 infant deaths globally each year. (Seepreeclampsia [dot] org.) In the United States, PE is responsible for100,000 premature births and 10,500 infant deaths each year at a cost ofroughly seven billion dollars (three billion dollars for maternaldisabilities and four billion dollars related to infant morbidity) everyyear to the health care system. Across the globe, PE and subsequenteclampsia are major contributors to maternal, fetal and neonatalmorbidity and mortality. Thus, PE represents a highly significant unmetpublic health need.

Although the root causes of PE have yet to be fully understood, it isnow well established that the maternal signs and symptoms ofhypertension, edema and proteinuria are caused by an excess ofanti-angiogenic proteins in the mother's bloodstream. Chief among theseare soluble fms-like tyrosine kinase 1 (sFLT1s) proteins. sFLT1s aretruncated forms of the membrane-bound vascular endothelial growth factor(VEGF) receptor FLT1 (also known as VEGFR1). They normally function tobuffer VEGF signaling. However, when sFLT1s are abnormally high in themother's circulatory system, they can interfere with her body's ownability to respond to VEGF. Among other functions, VEGF is required formaintenance of the hepatic sinusoidal vasculature and other fenestratedvascular beds in the body (Kamba, T. et al. VEGF-dependent plasticity offenestrated capillaries in the normal adult microvasculature. Americanjournal of physiology. Heart and circulatory physiology 290, H560-576(2006)). Breakdown of these structures impairs maternal kidney function,leading to hypertension, proteinuria and cerebral edema which areclassic features of PE and eclampsia (Young, B. C., Levine, R. J. &Karumanchi, S. A. Pathogenesis of preeclampsia. Annual review ofpathology 5, 173-192 (2010); Eremina, V. et al. Glomerular-specificalterations of VEGF-A expression lead to distinct congenital andacquired renal diseases. The Journal of clinical investigation 111,707-716 (2003); Eremina, V. et al. VEGF inhibition and renal thromboticmicroangiopathy. The New England journal of medicine 358, 1129-1136(2008)).

Pilot studies using an extracorporeal device to remove sFLT1 from thebloodstream of severely preeclamptic women has demonstrated thatlowering sFLT1 protein by just 30-40% in the maternal plasma can prolongPE pregnancies by 2 weeks without adverse consequences to the baby(Thadhani, R. et al. Pilot study of extracorporeal removal of solublefms-like tyrosine kinase 1 in preeclampsia. Circulation 124, 940-950(2011)). Moreover, animal studies support the hypothesis that targetingsFLT1 in PE may also lower the risk of neonatal respiratory problems andbronchopulmonary dysplasia, major complications of prematurity (Tang, J.R., Karumanchi, S. A., Seedorf, G., Markham, N. & Abman, S. H. Excesssoluble vascular endothelial growth factor receptor-1 in amniotic fluidimpairs lung growth in rats: linking preeclampsia with bronchopulmonarydysplasia. American journal of physiology. Lung cellular and molecularphysiology 302, L36-46 (2012)). Yet, while apheresis (blood washing) ishighly promising, it is unlikely to be applicable to all patients in allsituations. Especially in low resource settings, a more cost effectiveapproach with lower medical and general infrastructure requirements isdesperately needed. RNA silencing via RNAi is one such approach.

A broad range of human diseases, including cancer, infection andneurodegeneration, can be treated via the silencing of specific genesusing small oligonucleotides. ONTs (OligoNucleotide Therapeutics) are anew class of drugs, distinguished by targeting RNA or DNA directly, thusinterfering with a disease-causing gene at its root, before it canproduce the protein responsible for the disease phenotype. Advantages ofONTs over conventional drugs include ease of drug design based solely onbase-pairing rules, an ability to access targets previously considered“undruggable” and their promise of unprecedented specificity, potency,and duration of effect. In addition, pharmacokinetics, pharmacodynamicsand safety of ONTs is mostly defined by chemicalmodifications/formulation and is very similar between compound targetingdifferent genes, enabling multi-gene silencing and simple developmentdrugs targeting the same tissue (Videira, M., Arranja, A., Rafael, D. &Gaspar, R. Preclinical development of siRNA therapeutics: towards thematch between fundamental science and engineered systems. Nanomedicine:nanotechnology, biology, and medicine 10, 689-702 (2014); H. Younis etal. in A Comprehensive Guide to Toxicology in Preclinical DrugDevelopment. (ed. A. S. Faqi) 647-664 (Academic Press, 2013)).Significant effort in the last decade resulted in development of severaltypes of both chemically-modified and formulated oligonucleotides withclear clinical efficacy (Whitehead, K. A., Langer, R. & Anderson, D. G.Knocking down barriers: advances in siRNA delivery. Nature reviews. Drugdiscovery 8, 129-138 (2009)). Thus, ONTs represent a new and potentiallytransformative therapeutic paradigm. Nonetheless, their clinical utilityhas been hampered by limited tissue distribution. Systemicadministration has been generally limited to liver hepatocytes to date,with other tissues requiring local administration (de Fougerolles, A.,Vornlocher, H. P., Maraganore, J. & Lieberman, J. Interfering withdisease: a progress report on siRNA-based therapeutics. Nature reviews.Drug discovery 6, 443-453 (2007)).

One class of ONTs is siRNAs, small double-stranded oligonucleotidesconsisting of passenger (sense) and guide (antisense) strands. Uponcellular uptake, the guide strand is loaded into an RNA InducedSilencing Complex (RISC) capable of cleaving its complementary targetRNA. The numbers of loaded RISCs per cell sufficient to induce efficientand long-term gene silencing or RNA interference (RNAi) are estimated atapproximately 25-100 in vitro (Stalder, L. et al. The roughendoplasmatic reticulum is a central nucleation site of siRNA-mediatedRNA silencing. The EMBO journal 32, 1115-1127 (2013)) and approximately400 in vivo (Pei, Y. et al. Quantitative evaluation of siRNA delivery invivo. Rna 16, 2553-2563 (2010)). Typically, 10-100 ng/gram ofoligonucleotide delivered to a targeted tissue (Overhoff, M., Wunsche,W. & Sczakiel, G. Quantitative detection of siRNA and single-strandedoligonucleotides: relationship between uptake and biological activity ofsiRNA. Nucleic acids research 32, e170 (2004)) is adequate to generate asufficient number of active RISC complexes and induce silencing. LoadedRISCs have weeks long stability, resulting in prolonged gene silencing(3-6 weeks) from a single administration (Whitehead, K. A., Langer, R. &Anderson, D. G. Knocking down barriers: advances in siRNA delivery.Nature reviews. Drug discovery 8, 129-138 (2009)).

SUMMARY

The present invention in based in part on the discovery that mRNAisoforms encoding sFLT1 proteins contain sequences not found in mRNAencoding full length FLT1 (fl-FLT1) protein that can be targeted fordegradation, e.g., to treat PE, postpartum PE, eclampsia and/or HELLPsyndrome. Provided herein are novel oligonucleotide sequences (e.g.,small interfering RNAs (siRNAs)) that have been engineered toselectively decrease sFLT1 levels without affecting fl-FLT1 by bindingto one or more of the sequences that are not present in fl-FLT1, e.g.,one or more intronic regions of mRNA encoding one or more sFLT1proteins. It was discovered that the novel siRNAs described herein werepreferentially delivered to the placental trophoblasts (the cell typeresponsible for excess sFLT1 production) using systemic (i.e.,intravenous or subcutaneous) delivery to the mother without delivery tothe fetus. Therapeutic compounds and methods for treating one or moresymptoms of PE and/or postpartum PE and/or eclampsia and/or HELLPsyndrome are also provided.

In one aspect, a compound that binds to an intronic region of an mRNAencoding an sFLT1 protein, wherein the compound selectively inhibitsexpression of the sFLT1 protein in a cell or organism is provided.

In one embodiment, the compound comprises a single stranded (ss) RNAmolecule or a double stranded (ds) RNA molecule that is between 15 and35 bases in length. In one embodiment, the dsRNA molecule mediatesdegradation of the mRNA.

In one embodiment, the compound comprises a dsRNA having a sense strandand an antisense strand, wherein the antisense strand comprises a regionof complementarity which is substantially complementary to 5′CTCTCGGATCTCCAAATTTA 3′ (SEQ ID NO:1), 5′ CATCATAGCTACCATTTATT 3′ (SEQID NO:2), 5′ ATTGTACCACACAAAGTAAT 3′ (SEQ ID NO:3) or 5′GAGCCAAGACAATCATAACA 3′ (SEQ ID NO:4). In one embodiment, the region ofcomplementarity is complementary to at least 15, 16, 17 or 18 contiguousnucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. Inone embodiment, the region of complementarity contains no more than 3mismatches with SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.In one embodiment, the region of complementarity is fully complementaryto SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

In one embodiment, the dsRNA is blunt-ended. In one embodiment, thedsRNA comprises at least one single stranded nucleotide overhang. In oneembodiment, the dsRNA comprises naturally occurring nucleotides.

In one embodiment, the dsRNA comprises at least one modified nucleotide.In one embodiment, the modified nucleotide is chosen from the group of:a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, anucleotide comprising a 5′-phosphorothioate group, and a terminalnucleotide linked to a cholesteryl derivative. In one embodiment, themodified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. In one embodiment, thedsRNA comprises at least one 2′-O-methyl modified nucleotide, at leastone 2′-fluoro modified nucleotide, at least one nucleotide comprising a5′phosphorothioate group and a terminal nucleotide linked to acholesteryl derivative.

In one embodiment, the dsRNA has a 5′ end, a 3′ end and complementarityto a target, and comprises a first oligonucleotide and a secondoligonucleotide, wherein: (1) the first oligonucleotide comprises asequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3 and SEQ ID NO:4; (2) a portion of the first oligonucleotideis complementary to a portion of the second oligonucleotide; (3) thesecond oligonucleotide comprises alternating 2′-methoxy-ribonucleotidesand 2′-fluoro-ribonucleotides; (4) the nucleotides at positions 2 and 14from the 3′ end of the second oligonucleotide are2′-methoxy-ribonucleotides; and (5) the nucleotides of the secondoligonucleotide are connected via phosphodiester or phosphorothioatelinkages.

In one embodiment, the second oligonucleotide is linked to a hydrophobicmolecule at the 3′ end of the second oligonucleotide, e.g., an omega-3fatty acid. In another embodiment, the hydrophobic molecule isdocosanoic acid (DCA), docosahexaenoic acid (DHA),lysophosphatidylcholine esterified DHA (g2-DHA, also known as PC-DHA) oreicosapentaenoic acid (EPA).

In one embodiment, the linkage between the second oligonucleotide andthe hydrophobic molecule comprises polyethylene glycol or triethyleneglycol.

In one embodiment, the nucleotides at positions 1 and 2 from the 3′ endof second oligonucleotide are connected to adjacent nucleotides viaphosphorothioate linkages.

In one embodiment, the nucleotides at positions 1 and 2 from the 3′ endof second oligonucleotide, and the nucleotides at positions 1 and 2 fromthe 5′ end of second oligonucleotide, are connected to adjacentribonucleotides via phosphorothioate linkages.

In one embodiment, expression of the sFLT1 protein in the cell ororganism is reduced from about 30% to about 50%. In one embodiment,expression of the sFLT1 protein in the cell or organism is reduced fromabout 30% to about 40%.

In one aspect, a method for inhibiting expression of one or more sFLT1proteins in a cell is provided. The method includes the steps of (a)introducing into the cell one or more compounds that bind to an intronicregion of one or more mRNAs encoding one or more sFLT1 proteins, and (b)maintaining the cell produced in step (a) for a time sufficient toinhibit expression of the one or more sFLT1 s proteins in the cell.

In one embodiment, the one or more compounds are one or more dsRNAs thatmediate degradation of the one or more mRNAs. In one embodiment, acompound is a dsRNA having a sense strand and an antisense strand,wherein the antisense strand comprises a region of complementarity whichis substantially complementary to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,and/or SEQ ID NO:4. In one embodiment, one or more dsRNAs are eachbetween 15 and 30 base pairs in length.

In one embodiment, expression of one or more sFLT1 proteins is reducedfrom about 30% to about 50%. In one embodiment, expression of one ormore sFLT1 proteins is reduced from about 30% to about 40%.

In one aspect, an RNA molecule that is between 15 and 30 bases in lengthcomprising a region of complementarity which is substantiallycomplementary to SEQ ID NO: 1, wherein the RNA molecule targets one orboth of an intronic region of sFLT-i13 short and an intronic region ofsFLT-i13 long is provided.

In one embodiment, the RNA is dsRNA having a sense strand and anantisense strand, wherein the antisense strand comprises the region ofcomplementarity.

In one aspect, an RNA molecule that is between 15 and 30 bases in lengthcomprising a region of complementarity which is substantiallycomplementary to SEQ ID NO:2, wherein the RNA molecule targets one orboth of an intronic region of sFLT-i15a (also known as sFLT-e15a) isprovided.

In one embodiment, the RNA is dsRNA having a sense strand and anantisense strand, wherein the antisense strand comprises the region ofcomplementarity.

In one aspect, a therapeutic compound is provided that binds to anintronic region of one or more mRNAs encoding one or more sFLT1proteins, wherein the therapeutic compound selectively reducesexpression of the one or more sFLT1 proteins, and wherein thetherapeutic compound reduces one or more symptoms of PE, postpartum PE,eclampsia or HELLP syndrome when administered to a subject in needthereof.

In one embodiment, the one or more sFLT1 proteins are selected from thegroup consisting of sFLT1-i13 short, sFLT1-i13 long and sFlt1-i15a.

In one embodiment, the therapeutic compound comprises a first and asecond oligonucleotide sequence, wherein the first oligonucleotidesequence binds an intronic region of one or both of sFLT1-i13 short andsFLT1-i13 long, and the second oligonucleotide sequence binds anintronic region of sFlt1-i15a. In one embodiment, the first and secondoligonucleotide sequences are single stranded RNA (ssRNA) or doublestranded RNA (dsRNA).

In one embodiment, a therapeutic compound is provided comprising a firstdsRNA comprising a first sense strand and a first antisense strand and asecond dsRNA comprising a second sense strand and a second antisensestrand, wherein the first antisense strand comprises a first region ofcomplementarity which is substantially complementary to SEQ ID NO:1 andthe second antisense strand comprises a second region of complementaritywhich is substantially complementary to SEQ ID NO:2. In one embodiment,each dsRNA is between 15 and 30 base pairs in length. In one embodiment,the first region of complementarity is complementary to at least 15contiguous nucleotides of SEQ ID NO: 1, and the second region ofcomplementarity is complementary to at least 15 contiguous nucleotidesof SEQ ID NO:2. In one embodiment, the first region of complementaritycontains no more than 3 mismatches with SEQ ID NO: 1, and the secondregion of complementarity contains no more than 3 mismatches with SEQ IDNO:2. In one embodiment, the first region of complementarity is fullycomplementary to SEQ ID NO: 1, and the second region of complementarityis fully complementary to SEQ ID NO:2.

In one embodiment, each dsRNA comprises at least one single strandednucleotide overhang.

In one embodiment, each dsRNA comprises at least one modifiednucleotide. In one embodiment, the modified nucleotide is chosen fromthe group of: a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative. In oneembodiment, a modified nucleotide is chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. In one embodiment, a dsRNAcomprises at least one 2′-O-methyl modified nucleotide, at least one2′-fluoro modified nucleotide, at least one nucleotide comprising a5′phosphorothioate group and a terminal nucleotide linked to acholesteryl derivative.

In one embodiment, each dsRNA comprises a 5′ end, a 3′ end andcomplementarity to a target, wherein (1) the oligonucleotide comprisesalternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides;(2) the nucleotides at positions 2 and 14 from the 5′ end are not2′-methoxy-ribonucleotides; (3) the nucleotides are connected viaphosphodiester or phosphorothioate linkages; and (4) the nucleotides atpositions 1-6 from the 3′ end, or positions 1-7 from the 3′ end, areconnected to adjacent nucleotides via phosphorothioate linkages.

In one aspect, a pharmaceutical composition is provided. The compositionincludes a first dsRNA comprising a first sense strand and a firstantisense strand, wherein the first antisense strand comprises a regionof complementarity which is substantially complementary to SEQ ID NO:1,and wherein the first antisense strand selectively targets one or bothof an intronic region of sFLT-i13 short and an intronic region ofsFLT-i13 long; a second dsRNA comprising a second sense strand and asecond antisense strand, wherein the second antisense strand comprises aregion of complementarity which is substantially complementary to SEQ IDNO:2, and wherein the second antisense strand selectively targets anintronic region of sFLT-i15a; and a pharmaceutically acceptable carrier.

In one embodiment, a method of treating or managing PE, eclampsia orHELLP syndrome comprising administering to a subject in need of suchtreatment or management a therapeutically effective amount of apharmaceutical composition described herein is provided. In oneembodiment, the pharmaceutical composition is administered intravenouslyor subcutaneously. In one embodiment, sFLT1 protein expression isreduced in the subject by about 30% to about 50%. In one embodiment,sFLT1 protein expression is reduced in the subject by about 30% to about40%.

In one aspect, a method of treating one or more symptoms of PE,eclampsia or HELLP syndrome in a subject in need thereof is provided.The method includes administering to the subject a therapeutic compoundthat binds to an intronic region of one or more mRNAs encoding one ormore sFLT1 proteins, wherein the therapeutic compound reduces expressionof the one or more sFLT1 proteins.

In one embodiment, the one or more sFLT1 proteins are selected from thegroup consisting of sFLT1-i13 short, sFLT1-i13 long and sFlt1-i15a.

In one embodiment, the therapeutic compound comprises a first and asecond oligonucleotide sequence, wherein the first oligonucleotidesequence binds an intronic region of one or both of sFLT1-i13 short andsFLT1-i13 long, and the second oligonucleotide sequence binds anintronic region of sFlt1-i15a. In one embodiment, the first and secondoligonucleotide sequences are ssRNA or dsRNA.

In one embodiment, a therapeutic compound is provided comprising a firstdsRNA comprising a first sense strand and a first antisense strand and asecond dsRNA comprising a second sense strand and a second antisensestrand, wherein the first antisense strand comprises a first region ofcomplementarity which is substantially complementary to SEQ ID NO:1 andthe second antisense strand comprises a second region of complementaritywhich is substantially complementary to SEQ ID NO:2. In one embodiment,each dsRNA is between 15 and 30 base pairs in length. In one embodiment,the first region of complementarity is complementary to at least 15contiguous nucleotides of SEQ ID NO: 1, and the second region ofcomplementarity is complementary to at least 15 contiguous nucleotidesof SEQ ID NO:2. In one embodiment, the first region of complementaritycontains no more than 3 mismatches with SEQ ID NO: 1, and the secondregion of complementarity contains no more than 3 mismatches with SEQ IDNO:2. In one embodiment, the first region of complementarity is fullycomplementary to SEQ ID NO: 1, and the second region of complementarityis fully complementary to SEQ ID NO:2.

In one embodiment, each dsRNA comprises at least one single strandednucleotide overhang.

In one embodiment, each dsRNA comprises at least one modifiednucleotide. In one embodiment, the modified nucleotide is chosen fromthe group of: a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a nucleotide comprising a 5′-phosphorothioate group, and aterminal nucleotide linked to a cholesteryl derivative. In oneembodiment, a modified nucleotide is chosen from the group of: a2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide,a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide,2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate,and a non-natural base comprising nucleotide. In one embodiment, a dsRNAcomprises at least one 2′-O-methyl modified nucleotide, at least one2′-fluoro modified nucleotide, at least one nucleotide comprising a5′phosphorothioate group and a terminal nucleotide linked to acholesteryl derivative.

In one aspect, a pharmaceutical composition is provided. Thepharmaceutical composition includes a first dsRNA comprising a firstsense strand and a first antisense strand, wherein the first antisensestrand comprises a region of complementarity which is substantiallycomplementary to SEQ ID NO:1, and wherein the first antisense strandtargets one or both of an intronic region of sFLT-i13 short and anintronic region of sFLT-i13 long, a second dsRNA comprising a secondsense strand and a second antisense strand, wherein the second antisensestrand comprises a region of complementarity which is substantiallycomplementary to SEQ ID NO:2, and wherein the second antisense strandtargets an intronic region of sFLT-i15a, and a pharmaceuticallyacceptable carrier.

In one embodiment, a method of treating or managing PE, eclampsia orHELLP syndrome comprising administering to a subject in need of suchtreatment or management a therapeutically effective amount of thepharmaceutical composition described herein is provided.

In one embodiment, the pharmaceutical composition is administeredintravenously or subcutaneously.

In one embodiment, sFLT1 protein expression is reduced in the subject byabout 30% to about 50%. In one embodiment, sFLT1 protein expression isreduced in the subject by about 30% to about 40%.

In one aspect, a method of treating one or more symptoms of anangiogenic disorder in a subject in need thereof is provided, comprisingadministering to the subject any compound described herein.

In one aspect, a method of treating one or more symptoms of PE,eclampsia or HELLP syndrome in a subject in need thereof is provided,comprising administering to the subject any compound described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings.

FIGS. 1A-IC depict a PolyAdenylation Site Sequencing (PAS-Seq) analysisof sFLT1 isoform expression in preeclamptic and normal placentas. (A)Schematically depicts Receptor Tyrosine Kinase (RTK) signalingmodulation by soluble decoys, which can be generated by polyadenylationin an intron upstream of the TransMembrane (TM) and kinase domains.(Adapted from Vorlova, S. et al. Induction of antagonistic soluble decoyreceptor tyrosine kinases by intronic polyA activation. Molecular cell43, 927-939 (2011).) (B) PAS-Seq identifies alternative FLT1polyadenylation sites. (C) Both i13 and i15 isoforms are overexpressedin preeclampsia. Total RNA was purified and analyzed by PAS-Seq (Heyer,E. E., Ozadam, H., Ricci, E. P., Cenik, C. & Moore, M. J. An optimizedkit-free method for making strand-specific deep sequencing librariesfrom RNA fragments. Nucleic Acids Res 43, e2 (2015)) from five normaland six preeclamptic human placentas. Note that sFLT1-i14 refers tosFLT1-i15a.

FIG. 2 depicts the results of a Peptide Nucleic Acid (PNA)-based assayfor detection of sFLT1-i13-2283 in mouse tissues. Tissues were lysed,debris separated by precipitation, PNA-guide strand duplex purified byHigh Performance Liquid chromatography (HPLC) (DNAPac P100, 50% water50% acetonitrile and salt gradient was 0 to 1M NaClO₄) a systematicscreening of unformulated hsiRNAs targeting sFlt1 mRNA was performed.

FIGS. 3A-3D depict hydrophobic siRNA structural/chemical composition,uptake and efficacy in primary human cytotrophoblasts (CTBs). (A)Schematically depicts hydrophobically modified and stabilized siRNAs(hsiRNAs) according to certain embodiments. sFlt1-i13-2283 hsiRNA andmatching NTC was added to CTBs at concentration shown. (B) Level ofsFLT1 protein was measured by ELISA (#MVR100, R&D systems) inconditioned culture medium after 72 h treatment. (C) depicts sFlt1-i13mRNA levels, and (D) depicts Flt1-FL mRNA levels that were measuredusing QuantiGene® (Affymetrix) at 72 hours, (n=3, mean+/−SD).UNT—untreated cells, NTC—non-targeting control with matching chemistry.

FIGS. 4A-4B depict hsiRNA efficiency of delivery to liver, kidney andplacenta. (A) A wild-type pregnant mouse (E15) was injected withCy3-sFLT1-2283-P2 (red) (10 mg/kg; IV via tail vein). Tissues were fixedafter 24 hours, processed and imaged at 10× and 63× on a Leica tilingfluorescent microscope; nuclei stained with DAPI (blue). (B) Showstissue distribution of sFLT1-2283 (40 mg/kg) 5 days post injectionanalyzed by PNA assay (n=7, mean+SEM).

FIG. 5 depicts histological evaluation of hsiRNA distribution in mouseplacenta. A wild-type pregnant mouse (E15) was injected withCy3-sFLT1-2283-P2 (red) (10 mg/kg; IV via tail vein). Tissues were fixedafter 24 hours, processed and stained with HE, and then imaged at 20× ona Leica fluorescent microscope. Fm—Fetal membrane; mV—maternal vessel;L—labyrinth; Jz—junctional zone; D—decidua.

FIGS. 6A-6C depict the identification and validation of functionalhsiRNA compounds targeting i13 and i15 sFlt1 isoforms. (A) Schematicallyrepresents the exon-intron structure of sFLT1 i13 and i15 isoforms. (B)Depicts sFLT1 i13 and i15 mRNA sequences (SEQ ID NOS 6 and 111,respectively, in order of appearance). Locations of leading hsiRNAs hitsare indicated (red lines). Stop codons are shown in red. (C) depictshsiRNA targeting of sFLT1-i13 and sFlt1-i15a. FIG. 6(C) discloses the“Targeting region” sequences as SEQ ID NOS 112-115 and the“Oligonucleotide” sequences as SEQ ID NOS 116-123, all respectively, inorder of appearance. Chemical modifications are as follows:P—5′-phosphate; f—2′fluoro; m—2′O-methyl; #—phosphorothioate. Note thatsFLT1-i14 refers to sFLT1-i15a.

FIGS. 7A-7D depict efficient placental delivery and silencing of sFLT1in a wild-type mouse pregnancy model. (A) A wild-type pregnant mouse(E15) was injected with Cy3-sFLT1-2283-P2 (red) (10 mg/kg; IV via tailvein). Fetuses and their placentas were fixed after 24 hours, processed,and imaged on a Leica tiling fluorescent microscope; nuclei stained withDAPI (blue). (B) Depicts the sFlt1-i13 expression level in mouse tissues5 days after sFLT1-2283-P2 injection (2×20 mg/kg). sFLT1-i13 mRNA wasmeasured using QuantiGene® (Affymetrix), normalized to fl-FLT1 andpresented as percent of untreated control [n=3 (PBS); n=7(sFLT1-i13-2283), mean+SEM]. (C) Depicts a timeline of the experiment.(D) Depicts in vivo validation of sFLT1_2283/2519 (sFLT1-mix, 151111);CD1 mice via IV at 20 mg/kg, n=8).

FIGS. 8A-8C depict the impact of hsiRNA chemistry and route ofadministration on placental accumulation and distribution. (A) Awild-type pregnant mouse (E15) was injected with Cy3-sFLT1-2283 (red)(10 mg/kg; IV via tail vein). Placentas were fixed after 24 hours,processed, and imaged on a Leica tiling fluorescent microscope; nucleistained with DAPI (blue). (B) Depicts accumulation of sFLT1-i13-2283 (10mg/kg) after 24 hours, and analyzed by PNA assay (n=3, mean+SEM). (C)Schematically represents different modification patterns ofsFLT1-i13-2283 hsiRNA. P—5′-phosphate; Chol-teg—Cholesterol-teg linker;white spheres—RNA; black spheres—2′-O-methyl; grey spheres—2′-Fluoro;red spheres—phosphorothioate. Note that sFLT1-i14 refers to sFLT1-i15a.

FIGS. 9A-9B depict sFlt1 therapy in mice. (A) Show that sFlt1 therapy inmice induces hypertension (measured using radiotelemetry in consciousmice) and glomerular endotheliosis in the kidney (swollen glomeruli andcapillary occlusions). (B) Depicts Doppler ultrasound studies during anormal mouse pregnancy at late gestation to evaluate umbilical flow. Awaveform was obtained showing the Peak Systolic Velocity (PSV).

FIG. 10 depicts a flow chart showing steps for developing therapeutics(e.g., therapeutic RNAs) for the treatment of preeclampsia and/oreclampsia.

FIG. 11 schematically depicts factors associated with preeclampsia.

FIGS. 12A-12C depict selective delivery of hsiRNA to the syncytialtrophoblast layer of the mouse placenta labyrinth. (A) Depicts aschematic from Maltepe et al. (J Clin Invest. (2010) 120(4):1016-1025.doi:10.1172/JCI41211). (B) Depicts trophoblast distribution afterintravenous administration of hsiRNA (63× magnification). (C) Depictstrophoblast distribution after subcutaneous administration of hsiRNA(63× magnification).

FIG. 13 depicts a list of all hits with efficacy in different chemicalscaffolds and unique sequences of I13 short, I13 long and I15a isoforms,which were targeted as described further herein. FIG. 13 discloses“AUCGAGGUCCGCG” as SEQ ID NO: 16, “Targeting Region (20 mer)” sequencesdisclosed as SEQ ID NOS 124-182, “Targeting Region (30 mer)” sequencesdisclosed as SEQ ID NOS 183-241, “Sense Naked” sequences disclosed asSEQ ID NOS 242-300 and “Guide 20mer” sequences disclosed as SEQ ID NOS301-359, “Sense P0” sequences disclosed as SEQ ID NOS 360-418, “GuideP0” sequences disclosed as SEQ ID NOS 419-477, “Sense P1” sequencesdisclosed as SEQ ID NOS 478-536, “Guide P1” sequences disclosed as SEQID NOS 537-595, “Sense P2” sequences disclosed as SEQ ID NOS 596-654 and“Guide P2” sequences disclosed as SEQ ID NOS 655-713, all respectively,in order of appearance.

FIG. 14 summarizes acceptable and ideal target product profiles andcomments on the potential for addressing these needs according tocertain exemplary embodiments.

FIG. 15 depicts a histological evaluation of hsiRNA distribution inmouse placental tissues post-subcutaneous (SC) and post-intravenous (IV)administration.

FIGS. 16A-16E depict efficient silencing of sFLT1 by hsiRNA in pregnantnice (CD1). (A) Depicts a timeline of the experiment. (B) DepictssFLT1-I13 mRNA expression in liver, kidney and placenta. (C) DepictssFLT1 protein levels as a function of time. (D) Depicts the percentageof the sFLT1-2283 injected dose present in liver, kidney, placental,spleen and fetal liver tissues at five days post-injection. (E) Depictsμg/g sFLT1-2283 present in liver, kidney, placental, spleen and fetalliver tissues at five days post-injection.

FIGS. 17A-17D depict efficient silencing of sFLT1 by hsiRNA in pregnantnice (CD1). (A) Depicts a timeline of the experiment. (B) DepictsmsFlt1-1 levels protein detected in plasma as a function of days intopregnancy. (C) Is a table of mother mouse weight gains and pup weightsand mortality data. (D) Depicts graphs showing AST and ALT levels at day19.

FIGS. 18A-18B depict hsiRNA stability in vivo. IV vs. SC, sFLT_2283P2(150403). (A) Depicts a timeline of the experiment. (B) Depicts hsiRNAlevels in the liver post-IV and post-SC administration.

FIG. 19A-19B depicts hsiRNA stability in vivo. (A) Depicts a timeline ofthe experiment. (B) Depicts hsiRNA at two hours, 24 hours and 120 hourspost-IV administration. sFLT1_2283P2 (#150624).

FIG. 20 depicts tissue distribution in liver of DHA-hsiRNA and g2DHA-hsiRNA conjugates. 10× magnification. (sFLT1_2283P2-DHA,sFLT1_2283P2-g2DHA.) Blue, nucleus (DAPI); red, hsiRNA (Cy3). Mouse E15,IV (tail vein) injection. hsiRNAs administered at 10 mg/kg, 24 hours.LEICA DM5500B.

FIG. 21 depicts tissue distribution in liver of DHA-hsiRNA andg2DHA-hsiRNA conjugates. 63× magnification. (sFLT1_2283P2-DHA,sFLT1_2283P2-g2DHA.) Blue, nucleus (DAPI); red, hsiRNA (Cy3). Mouse E15,IV (tail vein) injection. hsiRNAs administered at 10 mg/kg, 24 hours.LEICA DM5500B.

FIG. 22 depicts tissue distribution in kidney of DHA-hsiRNA andg2DHA-hsiRNA conjugates. 10× magnification. (sFLT1_2283P2-DHA,sFLT1_2283P2-g2DHA.) Blue, nucleus (DAPI); red, hsiRNA (Cy3). Mouse E15,IV (tail vein) injection. hsiRNAs administered at 10 mg/kg, 24 hours.LEICA DM5500B.

FIG. 23 depicts tissue distribution in kidney of DHA-hsiRNA andg2DHA-hsiRNA conjugates. 63× magnification. (sFLT1_2283P2-DHA,sFLT1_2283P2-g2DHA.) Blue, nucleus (DAPI); red, hsiRNA (Cy3). Mouse E15,IV (tail vein) injection. hsiRNAs administered at 10 mg/kg, 24 hours.LEICA DM5500B.

FIG. 24 depicts tissue distribution in placenta of DHA-hsiRNA andg2DHA-hsiRNA conjugates. 10× magnification. (sFLT1_2283P2-DHA,sFLT1_2283P2-g2DHA.) Blue, nucleus (DAPI); red, hsiRNA (Cy3). Mouse E15,IV (tail vein) injection. hsiRNAs administered at 10 mg/kg, 24 hours.LEICA DM5500B.

FIG. 25 depicts tissue distribution in placenta of DHA-hsiRNA andg2DHA-hsiRNA conjugates. 63× magnification. (sFLT1_2283P2-DHA,sFLT1_2283P2-g2DHA.) Blue, nucleus (DAPI); red, hsiRNA (Cy3). Mouse E15,IV (tail vein) injection. hsiRNAs administered at 10 mg/kg, 24 hours.LEICA DM5500B.

FIG. 26 depicts sFLT1 silencing mediated by DHA-hsiRNA in pregnant mice(CD1) as detected in liver, kidney and placental tissues.sFLT1_2283P2-g2DHA (150813).

FIGS. 27A-27D depict in vitro validation of sFLT1_2283/2519 (sFLT1-mix,1510025). (A) Depicts a dose response of candidate siRNAs targetingsFLt1 i13 shown by mRNA expression levels. (B) Depicts a dose responseof candidate siRNAs targeting sFLt1 e15a shown by mRNA expressionlevels. (C) Depicts aFLT1 i13 mRNA expression levels in the presence of2283 (circles) or 2283/2519 (blocks). (D) Depicts aFLT1 e15a mRNAexpression levels in the presence of 2519 (circles) or 2283/2519(blocks).

FIGS. 28A-28D depict in vitro validation of sFLT1_2283/2519 (sFLT1-mix,151111). (A) Depicts a dose response of candidate siRNAs targeting sFLt1i13 shown by mRNA expression levels. (B) Depicts a dose response ofcandidate siRNAs targeting sFLt1 e15a shown by mRNA expression levels.(C) Depicts aFLT1 i13 mRNA expression levels in the presence of 2283(circles), 2283/2519 LS (blocks) or 2283/2519 (diamonds). (D) DepictsaFLT1 e15a mRNA expression levels in the presence of 2519 (circles),2283/2519 LS (blocks) or 2283/2519 (diamonds).

FIG. 29 depicts soluble sFLT1 protein modulation in pregnant mice usingsingle injections of sFLT1 2283/2519 (10 mg/kg each). sFLT1 proteinlevels at day 14/day 17 are shown in the left graph. sFLT1 proteinlevels in the serum as a function of pregnancy days are shown in theright graph.

FIG. 30 depicts a schematic of a baboon (Papio hamadrysas) PE model forstudying sFLT1_i13_2283P2/sFLT1_e15a_2519P2 efficacy and safety usingwild-type baboons with PE induced via uteroplacental ischemia (UPI).

FIG. 31 depicts passive uptake of FM-hsiRNA^(sFLT1) in primarytrophoblasts effective to decrease sFLT1 i13 mRNA expression.

FIG. 32 depicts systemic delivery of FM-hsiRNA^(sFLT1) (right twocolumns) relative to non-fully modified hsiRNA^(sFLT1) (left twocolumns) in liver, kidney and spleen tissues.

FIG. 33 depicts systemic delivery of FM-hsiRNAs in liver, kidney,spleen, skin and fat tissues after subcutaneous (SC) injection.

FIG. 34 depicts a PNA-based assay for guide strand quantification invivo.

FIGS. 35A-35F depict robust FM-hsiRNA^(sFLT1) delivery and efficacy inliver, kidney and spleen tissues in vivo after IV or SC administration.(A) Depicts ng/mg hsiRNA levels per tissue post-IV administration of 10mg/kg at t=24 hours. (B) Depicts ng/mg hsiRNA levels per tissue post-SCadministration of 10 mg/kg at t=24 hours. (C) Depicts ng/mg hsiRNAlevels per tissue post-IV administration of 2×20 mg/kg at t=120 hours.(D) Depicts ng/mg hsiRNA levels per tissue post-IV administration of2×15 mg/kg at t=120 hours. (E) Depicts sFLT1 mRNA expression post-IVadministration of 2×20 mg/kg at t=120 hours. (F) Depicts sFLT1 mRNAexpression post-IV administration of 2×20 mg/kg at t=120 hours.

FIG. 36 depicts an sFLT_2283/2519 hsiRNA mix according to particularlypreferred embodiments of the invention (SEQ ID NOS 8-11, respectively,in order of appearance). The species depicted in this drawing can be apharmaceutically acceptable salt, as the P—OH and P—SH would bedeprotonated.

FIG. 37 depicts data from a baboon PE model showing stabilization ofblood pressure in the animal.

FIG. 38 depicts data from a baboon PE model showing a decrease of bloodpressure in the animal.

FIG. 39 depicts exemplary sFLT1-2283/2519 dsRNAs conjugated tocholesterol. R1=5′-E-VP-mU, C₁₂H₁₈N₂O₉P₂S, Molecular Weight: 428.29,R2=3′-cholesterol, C₂₇H₄₆O, Molecular Weight: 386.66, connected by alinker defined as L.

(SEQ ID NO: 8) R¹═A═A-A-U-U-U-G-G-A-G-A-U-C═C═G═A═G═A═G, (SEQ ID NO: 9)R¹-A═U═U-U-A-A-A-C-C-U-C-U-A═G═G, (SEQ ID NO: 10)R¹═A═U-A-A-A-U-G-G-U-A-G-C-U═A═U═G═A═U═G, (SEQ ID NO: 11)R²-A═U═A-U-U-U-A-C-C-A-U-C-G═A═U.

FIG. 40 depicts exemplary sFLT1-2283/2519 dsRNAs conjugated to aphosphatidylcholine derivative of DHA (PC-DHA). R¹=5′-E-VP-mU,C₁₂H₁₈N₂O₉P₂S, Molecular Weight: 428.29. R2=3′-PC-DHA, C₃₈H₆₆N₃O₈P+,Molecular Weight: 723.93, connected by a linker defined as L.

(SEQ ID NO: 12) R¹═A═A-A-U-U-U-G-G-A-G-A-U-C═C═G═A═G═A═G,(SEQ ID NO: 13) R²-A═U═U-U-A-A-A-C-C-U-C-U-A═G═G, (SEQ ID NO 14)R¹═A═U-A-A-A-U-G-G-U-A-G-C-U═A═U═G═A═U═G, (SEQ ID NO: 15)R²-A═U═A-U-U-U-A-C-C-A-U-C-G═A═U.

FIG. 41 depicts examples of internucleotide linkages of R³. R³ is aninternucleotide bond between the first two nucleotides at the 5′ or 3′ends of any given oligonucleotide strand can be stabilized with themoieties depicted in this figure.

FIG. 42 depicts examples of internucleotide linkages of L².

FIG. 43 depicts examples of nucleosides of X¹, X², X³, and X⁴.

DETAILED DESCRIPTION

Novel angiogenic targets (e.g., PE target sequences, e.g., intronsequences of sFLT1 mRNAs) are provided. Also provided are novel siRNAsthat selectively target intronic regions of mRNAs encoding angiogenictargets (e.g., sFLT1 proteins). Methods of treating angiogenicdisorders, e.g., PE, postpartum PE, eclampsia and/or HELLP, are alsoprovided.

Generally, nomenclature used in connection with cell and tissue culture,molecular biology, immunology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art. The methods and techniquesprovided herein are generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification unless otherwise indicated. Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications, as commonly accomplished in the art or as describedherein. The nomenclature used in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well-known and commonly used in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art. In the event of any latent ambiguity,definitions provided herein take precedent over any dictionary orextrinsic definition. Unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including,” as well as other forms, such as “includes”and “included,” is not limiting.

So that the invention may be more readily understood, certain terms arefirst defined.

By “alteration” is meant a change (increase or decrease) in theexpression levels of a gene, mRNA or polypeptide as detected by standardart known methods such as those described herein. As used herein, anincrease or decrease includes a 10% change in expression levels, a 25%change, a 40% change, or a 50% or greater change in expression levels.In certain embodiments, an increase or decrease is a change inexpression levels of between about 30% and about 50% or between about30% and about 40%. “Alteration” can also indicate a change (increase ordecrease) in the biological activity of any of the mRNAs or polypeptidesof the invention (e.g., sFlt1 (e.g., sFlt1-i13 short, sFlt1-i13 longand/or sFlt1-i15a (also known as sFlt1-e15a)). Examples of biologicalactivity for sFlt-1 include one or more clinical symptoms of PE oreclampsia. As used herein, an increase or decrease includes a 10% changein biological activity, preferably a 25% change, more preferably a 40%change, and most preferably a 50% or greater change in biologicalactivity. In certain preferred embodiments, an increase or decrease is achange in expression levels of between about 30% and about 50% orbetween about 30% and about 40%.

Certain embodiments of the invention are directed to the treatment ofone or more angiogenic disorders. By “treatment of an angiogenicdisorder” is meant use of an oligonucleotide (e.g., an siRNA) of theinvention in a pharmaceutical composition for the treatment of diseasesinvolving the physiological and pathological processes ofneovascularization, vasculogenesis and/or angiogenesis. As such, thesepharmaceutical compositions are useful for treating diseases, conditionsand disorders that require inhibition of neovascularization,vasculogenesis or angiogenesis, including but not limited to cancertumor growth and metastasis, neoplasm, ocular neovascularization(including macular degeneration, diabetic retinopathy, ischemicretinopathy, retinopathy of prematurity, choroidal neovascularization),rheumatoid arthritis, osteoarthritis, chronic asthma, septic shock,inflammatory diseases, synovitis, bone and cartilage destruction, pannusgrowth, osteophyte formation, osteomyelitis, psoriasis, obesity,haemangioma, Kaposi's sarcoma, atherosclerosis (includingatherosclerotic plaque rupture), endometriosis, warts, excess hairgrowth, scar keloids, allergic edema, dysfunctional uterine bleeding,follicular cysts, ovarian hyperstimulation, endometriosis,osteomyelitis, inflammatory and infectious processes (hepatitis,pneumonia, glumerulonephtritis), asthma, nasal polyps, transplantation,liver regeneration, leukomalacia, thyroiditis, thyroid enlargement,lymphoproliferative disorders, haematologic malignancies, vascularmalformations, pre-eclampsia, eclampsia and/or HELLP syndrome.

By “preeclampsia” (“PE”) is meant the multi-system disorder that ischaracterized by hypertension with proteinuria or edema, or both, andone or more of glomerular dysfunction, brain edema, liver edema, orcoagulation abnormalities due to pregnancy or the influence of a recentpregnancy. PE generally occurs after the 20th week of gestation. PE isgenerally defined as some combination of the following symptoms: (1) asystolic blood pressure (BP)>140 mmHg and a diastolic BP>90 mmHg after20 weeks gestation (generally measured on two occasions, 4-168 hoursapart), (2) new onset proteinuria (1+ by dipstick on urinalysis, >300 mgof protein in a 24-hour urine collection, or a single random urinesample having a protein/creatinine ratio >v0.3), and (3) resolution ofhypertension and proteinuria by 12 weeks postpartum.

Severe PE is generally defined as (1) a diastolic BP>110 mmHg (generallymeasured on two occasions, 4-168 hours apart) or (2) proteinuriacharacterized by a measurement of 3.5 g or more protein in a 24-hoururine collection or two random urine specimens with at least 3+ proteinby dipstick. In PE, hypertension and proteinuria generally occur withinseven days of each other. In severe PE, severe hypertension, severeproteinuria and HELLP syndrome (Hemolysis, Elevated Liver enzymes, LowPlatelets) or eclampsia can occur simultaneously or only one symptom ata time.

Occasionally, severe PE can lead to the development of seizures. Thissevere form of the syndrome is referred to as “eclampsia.” Eclampsia canalso include dysfunction or damage to several organs or tissues such asthe liver (e.g., hepatocellular damage, periportal necrosis) and thecentral nervous system (e.g., cerebral edema and cerebral hemorrhage).The etiology of the seizures is thought to be secondary to thedevelopment of cerebral edema and focal spasm of small blood vessels inthe kidney.

By “HELLP” syndrome is meant a group of symptoms that occur in pregnantwoman characterized by hemolysis, elevated liver enzymes, and lowplatelet count. HELLP syndrome is thought to be a variant of PE, but itmay be an entity of its own.

In certain aspects, PE includes postpartum PE. Postpartum PE is a rarecondition that occurs when a woman has high blood pressure and excessprotein in her urine soon after childbirth. Postpartum PE typicallydevelops within 48 hours of childbirth. However, postpartum PE sometimesdevelops up to six weeks after childbirth, which is known as latepostpartum PE. Signs and symptoms of postpartum PE and late postpartumPE are typically similar to those of PE that occurs during pregnancy andmay include one or any combination of the following: high blood pressure(i.e., 140/90 mm Hg or greater; proteinuria; severe headaches; changesin vision, including temporary loss of vision, blurred vision or lightsensitivity; swelling of the face and limbs; upper abdominal pain,usually under the ribs on the right side; nausea or vomiting; anddecreased urination; sudden weight gain, typically more than 2 pounds(0.9 kilogram) a week.

By “intrauterine growth retardation (IUGR)” is meant a syndromeresulting in a birth weight which is less that 10 percent of thepredicted fetal weight for the gestational age of the fetus. The currentWorld Health Organization criterion for low birth weight is a weightless than 2,500 grams (5 lbs. 8 oz.) or below the 10th percentile forgestational age according to U.S. tables of birth weight for gestationalage by race, parity, and infant sex (Zhang and Bowes, Obstet. Gynecol.86:200-208, 1995). These low birth weight babies are also referred to as“small for gestational age (SGA).” PE is a condition known to beassociated with IUGR or SGA.

Certain embodiments of the invention are directed to the treatment ofone or more kidney disorders. By “treatment of a kidney disorder” ismeant use of an oligonucleotide (e.g., an siRNA) of the invention in apharmaceutical composition for the treatment of diseases, conditions ordisorders associated with the kidney. Diseases, conditions or disordersassociated with the kidney include, but are not limited to, ChronicKidney Disease (CKD) (stages 1-5 with stage 1 being the mildest andusually causing few symptoms and stage 5 being a severe illness withpoor life expectancy if untreated (stage 5 CKD is often called end stagerenal disease, end stage renal failure, or end-stage kidney disease,chronic kidney failure or chronic renal failure), and Acute RenalFailure (ARF) (caused by traumatic injury with blood loss, suddenreduction of blood flow to the kidneys, damage to the kidneys fromsepsis, obstruction of urine flow, damage from certain drugs or toxins,pregnancy complications (e.g., eclampsia, PE and/or HELLP syndrome) andthe like).

Certain embodiments of the invention are directed to the treatment ofone or more liver disorders. By “treatment of a liver disorder” is meantuse of an oligonucleotide (e.g., an siRNA) of the invention in apharmaceutical composition for the treatment of diseases, conditions ordisorders associated with the liver. Diseases, conditions or disordersassociated with the liver include, but are not limited to, fascioliasis,hepatitis (e.g., viral hepatitis, alcoholic hepatitis autoimmunehepatitis, hereditary hepatitis and the like), alcoholic liver disease(including alcoholic fatty liver disease, alcoholic hepatitis, andalcoholic cirrhosis), non-alcoholic fatty liver disease,steatohepatitis, non-alcoholic cirrhosis, primary liver cancer (e.g.,hepatocellular carcinoma, cholangiocarcinoma, angiosarcoma,hemangiosarcoma and the like), primary biliary cirrhosis, primarysclerosing, centrilobular necrosis, Budd-Chiari syndrome,hemochromatosis, Wilson's disease, alpha 1-antitrypsin deficiency,glycogen storage disease type II, transthyretin-related hereditaryamyloidosis, Gilbert's syndrome, biliary atresia, alpha-1 antitrypsindeficiency, Alagille syndrome, progressive familial intrahepaticcholestasis, and the like.

By “therapeutic amount” is meant an amount that when administered to apatient suffering from PE or eclampsia is sufficient to cause aqualitative or quantitative reduction in the symptoms of PE or eclampsiaas described herein. A “therapeutic amount” can also mean an amount thatwhen administered to a patient or subject suffering from PE or eclampsiais sufficient to cause a reduction in the expression levels of one ormore sFLT1 proteins (e.g., one or more of FLT1-i13 short, sFLT1-i13 longand sFlt1-i15a) as measured by one or more of the assays describedherein.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as non-human primates or other animals suchas, e.g., bovine, equine, canine, ovine, feline, murine and the like.Included in this definition are pregnant, post-partum and non-pregnantmammals.

By “soluble FLT1 (sFLT1)” (also known as sVEGF-R1) is meant a solubleform of the FLT1 receptor that has sFLT1 biological activity (e.g.,e.g., sFlt1-i13 short, sFlt1-i13 long and/or sFlt1-i15a (also known assFlt1-e15a)). The biological activity of an sFLT1 polypeptide may beassayed using any standard method, for example, by assaying for one ormore clinical symptoms of PE, postpartum PE, eclampsia and/or HELLP, byassaying sFLT1 mRNA and/or protein levels, by assaying sFLT1 binding toVEGF and the like. sFLT1 proteins lack the transmembrane domain and thecytoplasmic tyrosine kinase domain of the FLT1 receptor. sFLT1 proteinscan bind to VEGF and PlGF bind with high affinity, but cannot induceproliferation or angiogenesis and are therefore functionally differentfrom the Flt-1 and KDR receptors. sFLT1 was initially purified fromhuman umbilical endothelial cells and later shown to be produced bytrophoblast cells in vivo. As used herein, sFlt-1 includes any sFlt-1family member or isoform, e.g., sFLT1-i13 (e.g., FLT1-i13 short and/orsFLT1-i13 long (sFLT1_v1), sFlt1-i15a (sFLT1_v2), sFLT1-e15a, sFLT1_v3,sFLT1_v4 and the like.

The sequence of the sFLT1-i13 short isoform is:

(SEQ ID NO: 5) GTGAGCACTGCAACAAAAAGGCTGTTTTCTCTCGGATCTCCAAATTTAAAAGCACAAGGAATGATTGTACCACACAAAGTAATGTAAAACATTAAAGGAC TCATTAAAAAGTAA.

The sequence of the sFLT1-i13 long isoform is:

(SEQ ID NO: 6) GAAGAAAGAAATTACAATCAGAGGTGAGCACTGCAACAAAAAGGCTGTTTTCTCTCGGATCTCCAAATTTAAAAGCACAAGGAATGATTGTACCACACAAAGTAATGTAAAACATTAAAGGACTCATTAAAAAGTAACAGTTGTCTCATATCATCTTGATTTATTGTCACTGTTGCTAACTTTCAGGCTCGGAGGAGATGCTCCTCCCAAAATGAGTTCGGAGATGATAGCAGTAATAATGAGACCCCCGGGCTCCAGCTCTGGGCCCCCCATTCAGGCCGAGGGGGCTGCTCCGGGGGGCCGACTTGGTGCACGTTTGGATTTGGAGGATCCCTGCACTGCCTTCTCTGTGTTTGTTGCTCTTGCTGTTTTCTCCTGCCTGATAAACAACAACTTGGGATGATCCTTTCCATTTTGATGCCAACCTCTTTTTATTTTTAA GCGGCGCCCTATAGT.

The sequence of the sFLT1-i15a (also known as sFlt1-e15a) isoform is:

(SEQ ID NO: 7) AACTGTATACATCAACGTCACCATCGTCATCGTCATCATCACCATTGTCATCATCATCATCATCGTCATCATCATCATCATCATAGCTATCATCATTATCATCATCATCATCATCATCATCATAGCTACCATTTATTGAAAACTATTATGTGTCAACTTCAAAGAACTTATCCTTTAGTTGGAGAGCCAAGACAATCATAACAATAACAAATGGCCGGGCATGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGTGGATCATTTGAGGTCAGGAGTCCAAGACCAGCCTGACCAAGATGGTGAAATGCTGTCTCTATTAAAAATACAAAATTAGCCAGGCATGGTGGCTCATGCCTGTAATGCCAGCTACTCGGGAGGCTGAGACAGGAGAATCACTTGAACCCAGGAGGCAGAGGTTGCAGGGAGCCGAGATCGTGTACTGCACTCCAGCCTGGGCAACAAGAGCGAAACTCCGTCTCAAAAAACAAATAAATAAATAAATAAATAAACAGACAAAATTCACTTTTTATTCTATT AAACTTAACATACATGCTAA.

sFLT1 protein levels can be measured by measuring the amount of free,bound (i.e., bound to growth factor), or total sFLT1 (bound+free). VEGFor PlGF levels are determined by measuring the amount of free PlGF orfree VEGF (i.e., not bound to sFLT1). One exemplary metric is[sFLT1/(VEGF+PlGF)], also referred to as the PE anti-angiogenic index(PAAI).

By “pre-eclampsia anti-angiogenesis index (PAAI)” is meant the ratio ofsFLT1/VEGF+PlGF used as an indicator of anti-angiogenic activity. A PAAIgreater than 20 is considered to be indicative of PE or risk of PE.

By “vascular endothelial growth factor (VEGF)” is meant a mammaliangrowth factor that is homologous to the growth factor defined in U.S.Pat. Nos. 5,332,671; 5,240,848; 5,194,596; and Charnock-Jones et al.(Biol. Reproduction, 48: 1120-1128, 1993), and has VEGF biologicalactivity. VEGF exists as a glycosylated homodimer and includes at leastfour different alternatively spliced isoforms. The biological activityof native VEGF includes the promotion of selective growth of vascularendothelial cells or umbilical vein endothelial cells and induction ofangiogenesis. As used herein, VEGF includes any VEGF family member orisoform (e.g. VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF189, VEGF165,or VEGF 121). In certain embodiments, VEGF is the VEGF121 or VEGF 165isoform (Tischer et al., J. Biol. Chem. 266, 11947-11954, 1991; Neufedet al. Cancer Metastasis 15:153-158, 1996), which is described in U.S.Pat. Nos. 6,447,768; 5,219,739; and 5,194,596, hereby incorporated byreference. Also included are mutant forms of VEGF such as theKDR-selective VEGF and Flt-selective VEGF described in Gille et al. (J.Biol. Chem. 276:3222-3230, 2001). VEGF includes human forms and caninclude other animal forms of VEGF (e.g. mouse, rat, dog, chicken or thelike).

By “placental growth factor (PlGF)” is meant a mammalian growth factorthat is homologous to the protein defined by GenBank accession numberP49763 and that has PlGF biological activity. PlGF is a glycosylatedhomodimer belonging to the VEGF family and can be found in two distinctisoforms through alternative splicing mechanisms. PlGF is expressed bycyto- and syncytiotrophoblasts in the placenta and PlGF biologicalactivities include induction of proliferation, migration, and activationof endothelial cells, particularly trophoblast cells.

By “trophoblast” is meant the mesectodermal cell layer covering theblastocyst that erodes the uterine mucosa and through which the embryoreceives nourishment from the mother. Trophoblast cells contribute tothe formation of the placenta.

The term “nucleoside” refers to a molecule having a purine or pyrimidinebase covalently linked to a ribose or deoxyribose sugar. Exemplarynucleosides include adenosine, guanosine, cytidine, uridine andthymidine. Additional exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine,2N-methylguanosine and 2,2N,N-dimethylguanosine (also referred to as“rare” nucleosides). The term “nucleotide” refers to a nucleoside havingone or more phosphate groups joined in ester linkages to the sugarmoiety. Exemplary nucleotides include nucleoside monophosphates,diphosphates and triphosphates. The terms “polynucleotide” and “nucleicacid molecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester linkage between 5′ and3′ carbon atoms.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refersto a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30,or more ribonucleotides). The term “DNA” or “DNA molecule” or“deoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively). RNA can bepost-transcriptionally modified. DNA and RNA can also be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNAthat specifies the amino acid sequence of one or more polypeptidechains. This information is translated during protein synthesis whenribosomes bind to the mRNA.

As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to an RNA (orRNA analog) comprising between about 10-50 nucleotides (or nucleotideanalogs) which is capable of directing or mediating RNA interference.Preferably, a siRNA comprises between about 15-30 nucleotides ornucleotide analogs, more preferably between about 16-25 nucleotides (ornucleotide analogs), even more preferably between about 18-23nucleotides (or nucleotide analogs), and even more preferably betweenabout 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22nucleotides or nucleotide analogs). The term “short” siRNA refers to asiRNA comprising about 21 nucleotides (or nucleotide analogs), forexample, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to asiRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26nucleotides. Short siRNAs may, in some instances, include fewer than 19nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shortersiRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, insome instances, include more than 26 nucleotides, provided that thelonger siRNA retains the ability to mediate RNAi absent furtherprocessing, e.g., enzymatic processing, to a short siRNA.

The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of positions of thenucleotide which may be derivatized include the 5 position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine,5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyluridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromoguanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotideanalogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- andN-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwiseknown in the art) nucleotides; and other heterocyclically modifiednucleotide analogs such as those described in Herdewijn, AntisenseNucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example the 2′ OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR,or OR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, alkenyl,alkynyl, aryl, etc. Other possible modifications include those describedin U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vitro.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. The term “RNA analog” refers to anpolynucleotide (e.g., a chemically synthesized polynucleotide) having atleast one altered or modified nucleotide as compared to a correspondingunaltered or unmodified RNA but retaining the same or similar nature orfunction as the corresponding unaltered or unmodified RNA. As discussedabove, the oligonucleotides may be linked with linkages which result ina lower rate of hydrolysis of the RNA analog as compared to an RNAmolecule with phosphodiester linkages. For example, the nucleotides ofthe analog may comprise methylenediol, ethylene diol, oxymethylthio,oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate,and/or phosphorothioate linkages. Preferred RNA analogues include sugar-and/or backbone-modified ribonucleotides and/or deoxyribonucleotides.Such alterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA). An RNA analog need only besufficiently similar to natural RNA that it has the ability to mediate(mediates) RNA interference.

As used herein, the term “RNA interference” (“RNAi”) refers to aselective intracellular degradation of RNA. RNAi occurs in cellsnaturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAiproceeds via fragments cleaved from free dsRNA which direct thedegradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of target genes.

An RNAi agent, e.g., an RNA silencing agent, having a strand which is“sequence sufficiently complementary to a target mRNA sequence to directtarget-specific RNA interference (RNAi)” means that the strand has asequence sufficient to trigger the destruction of the target mRNA by theRNAi machinery or process.

As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or“isolated siRNA precursor”) refers to RNA molecules which aresubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

As used herein, the term “RNA silencing” refers to a group ofsequence-specific regulatory mechanisms (e.g. RNA interference (RNAi),transcriptional gene silencing (TGS), post-transcriptional genesilencing (PTGS), quelling, co-suppression, and translationalrepression) mediated by RNA molecules which result in the inhibition or“silencing” of the expression of a corresponding protein-coding gene.RNA silencing has been observed in many types of organisms, includingplants, animals, and fungi.

The term “discriminatory RNA silencing” refers to the ability of an RNAmolecule to substantially inhibit the expression of a “first” or“target” polynucleotide sequence while not substantially inhibiting theexpression of a “second” or “non-target polynucleotide sequence,” e.g.,when both polynucleotide sequences are present in the same cell. Incertain embodiments, the target polynucleotide sequence corresponds to atarget gene, while the non-target polynucleotide sequence corresponds toa non-target gene. In other embodiments, the target polynucleotidesequence corresponds to a target allele, while the non-targetpolynucleotide sequence corresponds to a non-target allele. In certainembodiments, the target polynucleotide sequence is the DNA sequenceencoding the regulatory region (e.g. promoter or enhancer elements) of atarget gene. In other embodiments, the target polynucleotide sequence isa target mRNA encoded by a target gene.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

As used herein, the term “transgene” refers to any nucleic acidmolecule, which is inserted by artifice into a cell, and becomes part ofthe genome of the organism that develops from the cell. Such a transgenemay include a gene that is partly or entirely heterologous (i.e.,foreign) to the transgenic organism, or may represent a gene homologousto an endogenous gene of the organism. The term “transgene” also means anucleic acid molecule that includes one or more selected nucleic acidsequences, e.g., DNAs, that encode one or more engineered RNAprecursors, to be expressed in a transgenic organism, e.g., animal,which is partly or entirely heterologous, i.e., foreign, to thetransgenic animal, or homologous to an endogenous gene of the transgenicanimal, but which is designed to be inserted into the animal's genome ata location which differs from that of the natural gene. A transgeneincludes one or more promoters and any other DNA, such as introns,necessary for expression of the selected nucleic acid sequence, alloperably linked to the selected sequence, and may include an enhancersequence.

A gene “involved” in a disease or disorder includes a gene, the normalor aberrant expression or function of which effects or causes thedisease or disorder or at least one symptom of said disease or disorder.

The term “gain-of-function mutation” as used herein, refers to anymutation in a gene in which the protein encoded by said gene (i.e., themutant protein) acquires a function not normally associated with theprotein (i.e., the wild type protein) causes or contributes to a diseaseor disorder. The gain-of-function mutation can be a deletion, addition,or substitution of a nucleotide or nucleotides in the gene which givesrise to the change in the function of the encoded protein. In oneembodiment, the gain-of-function mutation changes the function of themutant protein (e.g., causes production of one or more sFLT1 proteins)or causes interactions with other proteins. In another embodiment, thegain-of-function mutation causes a decrease in or removal of normalwild-type protein, for example, by interaction of the altered, mutantprotein with said normal, wild-type protein.

As used herein, the term “target gene” is a gene whose expression is tobe substantially inhibited or “silenced.” This silencing can be achievedby RNA silencing, e.g., by cleaving the mRNA of the target gene ortranslational repression of the target gene. The term “non-target gene”is a gene whose expression is not to be substantially silenced. In oneembodiment, the polynucleotide sequences of the target and non-targetgene (e.g. mRNA encoded by the target (sFLT1) and non-target (flFLT1)genes) can differ by one or more nucleotides, e.g., at an intronicregion. In another embodiment, the target and non-target genes candiffer by one or more polymorphisms (e.g., Single NucleotidePolymorphisms or SNPs). In another embodiment, the target and non-targetgenes can share less than 100% sequence identity. In another embodiment,the non-target gene may be a homologue (e.g. an orthologue or paralogue)of the target gene.

A “target allele” is an allele (e.g., a SNP allele) whose expression isto be selectively inhibited or “silenced.” This silencing can beachieved by RNA silencing, e.g., by cleaving the mRNA of the target geneor target allele by a siRNA. The term “non-target allele” is a allelewhose expression is not to be substantially silenced. In certainembodiments, the target and non-target alleles can correspond to thesame target gene. In other embodiments, the target allele correspondsto, or is associated with, a target gene, and the non-target allelecorresponds to, or is associated with, a non-target gene. In oneembodiment, the polynucleotide sequences of the target and non-targetalleles can differ by one or more nucleotides. In another embodiment,the target and non-target alleles can differ by one or more allelicpolymorphisms (e.g., one or more SNPs). In another embodiment, thetarget and non-target alleles can share less than 100% sequenceidentity.

The term “polymorphism” as used herein, refers to a variation (e.g., oneor more deletions, insertions, or substitutions) in a gene sequence thatis identified or detected when the same gene sequence from differentsources or subjects (but from the same organism) are compared. Forexample, a polymorphism can be identified when the same gene sequencefrom different subjects are compared. Identification of suchpolymorphisms is routine in the art, the methodologies being similar tothose used to detect, for example, breast cancer point mutations.Identification can be made, for example, from DNA extracted from asubject's lymphocytes, followed by amplification of polymorphic regionsusing specific primers to said polymorphic region. Alternatively, thepolymorphism can be identified when two alleles of the same gene arecompared. In particular embodiments, the polymorphism is a singlenucleotide polymorphism (SNP).

A variation in sequence between two alleles of the same gene within anorganism is referred to herein as an “allelic polymorphism.” In certainembodiments, the allelic polymorphism corresponds to a SNP allele. Forexample, the allelic polymorphism may comprise a single nucleotidevariation between the two alleles of a SNP. The polymorphism can be at anucleotide within a coding region but, due to the degeneracy of thegenetic code, no change in amino acid sequence is encoded.Alternatively, polymorphic sequences can encode a different amino acidat a particular position, but the change in the amino acid does notaffect protein function. Polymorphic regions can also be found innon-encoding regions of the gene. In exemplary embodiments, thepolymorphism is found in a coding region of the gene or in anuntranslated region (e.g., a 5′ UTR or 3′ UTR) of the gene.

As used herein, the term “allelic frequency” is a measure (e.g.,proportion or percentage) of the relative frequency of an allele (e.g.,a SNP allele) at a single locus in a population of individuals. Forexample, where a population of individuals carry n loci of a particularchromosomal locus (and the gene occupying the locus) in each of theirsomatic cells, then the allelic frequency of an allele is the fractionor percentage of loci that the allele occupies within the population. Inparticular embodiments, the allelic frequency of an allele (e.g., an SNPallele) is at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40% ormore) in a sample population.

As used herein, the term “sample population” refers to a population ofindividuals comprising a statistically significant number ofindividuals. For example, the sample population may comprise 50, 75,100, 200, 500, 1000 or more individuals. In particular embodiments, thesample population may comprise individuals which share at least oncommon disease phenotype (e.g., a gain-of-function disorder) or mutation(e.g., a gain-of-function mutation).

As used herein, the term “heterozygosity” refers to the fraction ofindividuals within a population that are heterozygous (e.g., contain twoor more different alleles) at a particular locus (e.g., at a SNP).Heterozygosity may be calculated for a sample population using methodsthat are well known to those skilled in the art.

The phrase “examining the function of a gene in a cell or organism”refers to examining or studying the expression, activity, function orphenotype arising therefrom.

As used herein, the term “RNA silencing agent” refers to an RNA which iscapable of inhibiting or “silencing” the expression of a target gene. Incertain embodiments, the RNA silencing agent is capable of preventingcomplete processing (e.g., the full translation and/or expression) of amRNA molecule through a post-transcriptional silencing mechanism. RNAsilencing agents include small (<50 b.p.), noncoding RNA molecules, forexample RNA duplexes comprising paired strands, as well as precursorRNAs from which such small non-coding RNAs can be generated. ExemplaryRNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, anddual-function oligonucleotides as well as precursors thereof. In oneembodiment, the RNA silencing agent is capable of inducing RNAinterference. In another embodiment, the RNA silencing agent is capableof mediating translational repression.

As used herein, the term “rare nucleotide” refers to a naturallyoccurring nucleotide that occurs infrequently, including naturallyoccurring deoxyribonucleotides or ribonucleotides that occurinfrequently, e.g., a naturally occurring ribonucleotide that is notguanosine, adenosine, cytosine, or uridine. Examples of rare nucleotidesinclude, but are not limited to, inosine, 1-methyl inosine,pseudouridine, 5,6-dihydrouridine, ribothymidine, ²N-methylguanosine and^(2,2)N,N-dimethylguanosine.

The term “engineered,” as in an engineered RNA precursor, or anengineered nucleic acid molecule, indicates that the precursor ormolecule is not found in nature, in that all or a portion of the nucleicacid sequence of the precursor or molecule is created or selected by ahuman. Once created or selected, the sequence can be replicated,translated, transcribed, or otherwise processed by mechanisms within acell. Thus, an RNA precursor produced within a cell from a transgenethat includes an engineered nucleic acid molecule is an engineered RNAprecursor.

As used herein, the term “microRNA” (“miRNA”), also referred to in theart as “small temporal RNAs” (“stRNAs”), refers to a small (10-50nucleotide) RNA which are genetically encoded (e.g., by viral,mammalian, or plant genomes) and are capable of directing or mediatingRNA silencing. An “miRNA disorder” shall refer to a disease or disordercharacterized by an aberrant expression or activity of an miRNA.

As used herein, the term “dual functional oligonucleotide” refers to aRNA silencing agent having the formula T-L-μ, wherein T is an mRNAtargeting moiety, L is a linking moiety, and t is a miRNA recruitingmoiety. As used herein, the terms “mRNA targeting moiety,” “targetingmoiety,” “mRNA targeting portion” or “targeting portion” refer to adomain, portion or region of the dual functional oligonucleotide havingsufficient size and sufficient complementarity to a portion or region ofan mRNA chosen or targeted for silencing (i.e., the moiety has asequence sufficient to capture the target mRNA). As used herein, theterm “linking moiety” or “linking portion” refers to a domain, portionor region of the RNA-silencing agent which covalently joins or links themRNA.

As used herein, the term “antisense strand” of an RNA silencing agent,e.g., an siRNA or RNA silencing agent, refers to a strand that issubstantially complementary to a section of about 10-50 nucleotides,e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of thegene targeted for silencing. The antisense strand or first strand hassequence sufficiently complementary to the desired target mRNA sequenceto direct target-specific silencing, e.g., complementarity sufficient totrigger the destruction of the desired target mRNA by the RNAi machineryor process (RNAi interference) or complementarity sufficient to triggertranslational repression of the desired target mRNA.

The term “sense strand” or “second strand” of an RNA silencing agent,e.g., an siRNA or RNA silencing agent, refers to a strand that iscomplementary to the antisense strand or first strand. Antisense andsense strands can also be referred to as first or second strands, thefirst or second strand having complementarity to the target sequence andthe respective second or first strand having complementarity to saidfirst or second strand. miRNA duplex intermediates or siRNA-likeduplexes include a miRNA strand having sufficient complementarity to asection of about 10-50 nucleotides of the mRNA of the gene targeted forsilencing and a miRNA* strand having sufficient complementarity to forma duplex with the miRNA strand.

As used herein, the term “guide strand” refers to a strand of an RNAsilencing agent, e.g., an antisense strand of an siRNA duplex or siRNAsequence, that enters into the RISC complex and directs cleavage of thetarget mRNA.

As used herein, the term “asymmetry,” as in the asymmetry of the duplexregion of an RNA silencing agent (e.g., the stem of an shRNA), refers toan inequality of bond strength or base pairing strength between thetermini of the RNA silencing agent (e.g., between terminal nucleotideson a first strand or stem portion and terminal nucleotides on anopposing second strand or stem portion), such that the 5′ end of onestrand of the duplex is more frequently in a transient unpaired, e.g.,single-stranded, state than the 5′ end of the complementary strand. Thisstructural difference determines that one strand of the duplex ispreferentially incorporated into a RISC complex. The strand whose 5′ endis less tightly paired to the complementary strand will preferentiallybe incorporated into RISC and mediate RNAi.

As used herein, the term “bond strength” or “base pair strength” refersto the strength of the interaction between pairs of nucleotides (ornucleotide analogs) on opposing strands of an oligonucleotide duplex(e.g., an siRNA duplex), due primarily to H-bonding, van der Waalsinteractions, and the like between said nucleotides (or nucleotideanalogs).

As used herein, the “5′ end,” as in the 5′ end of an antisense strand,refers to the 5′ terminal nucleotides, e.g., between one and about 5nucleotides at the 5′ terminus of the antisense strand. As used herein,the “3′ end,” as in the 3′ end of a sense strand, refers to the region,e.g., a region of between one and about 5 nucleotides, that iscomplementary to the nucleotides of the 5′ end of the complementaryantisense strand.

As used herein the term “destabilizing nucleotide” refers to a firstnucleotide or nucleotide analog capable of forming a base pair withsecond nucleotide or nucleotide analog such that the base pair is oflower bond strength than a conventional base pair (i.e., Watson-Crickbase pair). In certain embodiments, the destabilizing nucleotide iscapable of forming a mismatch base pair with the second nucleotide. Inother embodiments, the destabilizing nucleotide is capable of forming awobble base pair with the second nucleotide. In yet other embodiments,the destabilizing nucleotide is capable of forming an ambiguous basepair with the second nucleotide.

As used herein, the term “base pair” refers to the interaction betweenpairs of nucleotides (or nucleotide analogs) on opposing strands of anoligonucleotide duplex (e.g., a duplex formed by a strand of a RNAsilencing agent and a target mRNA sequence), due primarily to H-bonding,van der Waals interactions, and the like between said nucleotides (ornucleotide analogs). As used herein, the term “bond strength” or “basepair strength” refers to the strength of the base pair.

As used herein, the term “mismatched base pair” refers to a base pairconsisting of non-complementary or non-Watson-Crick base pairs, forexample, not normal complementary G:C, A:T or A:U base pairs. As usedherein the term “ambiguous base pair” (also known as anon-discriminatory base pair) refers to a base pair formed by auniversal nucleotide.

As used herein, term “universal nucleotide” (also known as a “neutralnucleotide”) include those nucleotides (e.g. certain destabilizingnucleotides) having a base (a “universal base” or “neutral base”) thatdoes not significantly discriminate between bases on a complementarypolynucleotide when forming a base pair. Universal nucleotides arepredominantly hydrophobic molecules that can pack efficiently intoantiparallel duplex nucleic acids (e.g., double-stranded DNA or RNA) dueto stacking interactions. The base portions of universal nucleotidestypically comprise a nitrogen-containing aromatic heterocyclic moiety.

As used herein, the terms “sufficient complementarity” or “sufficientdegree of complementarity” mean that the RNA silencing agent has asequence (e.g. in the antisense strand, mRNA targeting moiety or miRNArecruiting moiety) which is sufficient to bind the desired target RNA,respectively, and to trigger the RNA silencing of the target mRNA.

As used herein, the term “translational repression” refers to aselective inhibition of mRNA translation. Natural translationalrepression proceeds via miRNAs cleaved from shRNA precursors. Both RNAiand translational repression are mediated by RISC. Both RNAi andtranslational repression occur naturally or can be initiated by the handof man, for example, to silence the expression of target genes.

Various methodologies of the instant invention include step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control,” referred to interchangeably herein as an“appropriate control.” A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing an RNA silencing agent of the invention into a cellor organism. In another embodiment, a “suitable control” or “appropriatecontrol” is a value, level, feature, characteristic, property, etc.determined in a cell or organism, e.g., a control or normal cell ororganism, exhibiting, for example, normal traits. In yet anotherembodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, etc.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and example are illustrative only and not intendedto be limiting.

In some embodiments, the RNA silencing agents of the invention aredesigned to target intronic regions in mRNA molecules encoding one ormore sFLT1 proteins.

The present invention targets one or more sFLT1 mRNAs and theircorresponding proteins. One strand of double-stranded RNA (siRNA)complements a target sequence within the sFLT1 mRNA. After introductionof siRNA into a subject or cell, the siRNA partially unwinds, binds toan intronic target region within the sFLT1 mRNA in a site-specificmanner, and activates an mRNA nuclease. This nuclease cleaves the sFLT1mRNA, thereby halting translation of the sFLT1 protein. Cells ridthemselves of partially digested mRNA, thus precluding translation, orcells digest partially translated proteins. In certain embodiments,sFLT1 protein expression is reduced in a subject or cell by about 30% to50%, or by about 30% to 40%.

In embodiments of the invention, RNA silencing agents of the inventionare capable of targeting one or more of the target sequences listed inFIG. 13. In certain exemplary embodiments, RNA silencing agents of theinvention are capable of targeting one or more of the target sequencesat one or more target sequences listed at gene positions selected fromthe group consisting of 2247, 2252, 2253, 2256, 2279, 2280, 2283, 2284,2286, 2293, 2294, 2295, 2304, 2313, 2318, 2321, 2322, 2324, 2326, 2332,2333, 2339, 2343, 2351, 2353, 2362, 2471, 2474, 2477, 2508, 2510, 2513,2518, 2519, 2525, 2528, 2556, 2561, 2572, 2574, 2576, 2577, 2580, 2582,2585, 2588 and 2590 of the human flt1 gene (as set forth at FIG. 13 andin the Table below). Particularly exemplary target sequences of thehumanflt1 gene can be found at positions 2283 (5′ CTCTCGGATCTCCAAATTTA3′ (SEQ ID NO:1)), 2519 (5′ CATCATAGCTACCATTTATT 3′ (SEQ ID NO:2)), 2318(5′ ATTGTACCACACAAAGTAAT 3′ (SEQ ID NO:3)) and 2585 (5′GAGCCAAGACAATCATAACA 3′ (SEQ ID NO:4)). (See FIGS. 6 and 13.) Genomicsequence for each target sequence can be found in, for example, thepublically available database maintained by the NCBI.

TABLE 1 Targeted hits with efficacy for sFLT-i13 short, sFLT1-i13 longand sFLT-i15a isoforms. Table 1 discloses “AUCGAGGUCCGCG” as SEQ ID NO: 16,“Targeting Region (20 mer)” sequences as SEQ ID NOS 16-63 and “Targeting Region (30 mer)”sequences as SEQ ID NOS 64-110, all respectively, in order of appearance.AUCGAGGUCCGCG Accession Number Position Targeting region (20 mer)Targeting Region (30 mer) sFLT1-i13 NM_001159920.1 2247AAUCAGAGGUGAGCACUGCA AUUACAAUCAGAGGUGAGCACUGCAACAAA sFLT1-i13NM_001159920.1 2252 GAGGUGAGCACUGCAACAAA AAUCAGAGGUGAGCACUGCAACAAAAAGGCsFLT1-i13 NM_001159920.1 2253 AGGUGAGCACUGCAACAAAAAUCAGAGGUGAGCACUGCAACAAAAAGGCU sFLT1-i13 NM_001159920.1 2256UGAGCACUGCAACAAAAAGG AGAGGUGAGCACUGCAACAAAAAGGCUGUU sFLT1-i13NM_001159920.1 2279 UUUUCUCUCGGAUCUCCAAA GGCUGUUUUCUCUCGGAUCUCCAAAUUUAAsFLT1-i13 NM_001159920.1 2280 UUUCUCUCGGAUCUCCAAAUGCUGUUUUCUCUCGGAUCUCCAAAUUUAAA sFLT1-i14 NM_001159920.2 2283CUCUCGGAUCUCCAAAUUUA GUUUUCUCUCGGAUCUCCAAAUUUAAAAGC sFLT1-i13NM_001159920.1 2284 UCUCGGAUCUCCAAAUUUAA UUUUCUCUCGGAUCUCCAAAUUUAAAAGCAsFLT1-i13 NM_001159920.1 2286 UCGGAUCUCCAAAUUUAAAAUUCUCUCGGAUCUCCAAAUUUAAAAGCACA sFLT1-i13 NM_001159920.1 2293UCCAAAUUUAAAAGCACAAG GGAUCUCCAAAUUUAAAAGCACAAGGAAUG sFLT1-i13NM_001159920.1 2294 CCAAAUUUAAAAGCACAAGG GAUCUCCAAAUUUAAAAGCACAAGGAAUGAsFLT1-i13 NM_001159920.1 2295 CAAAUUUAAAAGCACAAGGAAUCUCCAAAUUUAAAAGCACAAGGAAUGAU sFLT1-i13 NM_001159920.1 2304AAGCACAAGGAAUGAUUGUA UUUAAAAGCACAAGGAAUGAUUGUACCACA sFLT1-i13NM_001159920.1 2313 GAAUGAUUGUACCACACAAA ACAAGGAAUGAUUGUACCACACAAAGUAAUsFLT1-i13 NM_001159920.1 2318 AUUGUACCACACAAAGUAAUGAAUGAUUGUACCACACAAAGUAAUGUAAA sFLT1-i13 NM_001159920.1 2321GUACCACACAAAGUAAUGUA UGAUUGUACCACACAAAGUAAUGUAAAACA sFLT1-i13NM_001159920.1 2322 UACCACACAAAGUAAUGUAA GAUUGUACCACACAAAGUAAUGUAAAACAUsFLT1-i13 NM_001159920.1 2324 CCACACAAAGUAAUGUAAAAUUGUACCACACAAAGUAAUGUAAAACAUUA sFLT1-i13 NM_001159920.1 2326ACACAAAGUAAUGUAAAACA GUACCACACAAAGUAAUGUAAAACAUUAAA sFLT1-i13NM_001159920.1 2332 AGUAAUGUAAAACAUUAAAG CACAAAGUAAUGUAAAACAUUAAAGGACUCsFLT1-i13 NM_001159920.1 2333 GUAAUGUAAAACAUUAAAGGACAAAGUAAUGUAAAACAUUAAAGGACUCA sFLT1-i13 NM_001159920.1 2339UAAAACAUUAAAGGACUCAU UAAUGUAAAACAUUAAAGGACUCAUUAAAA sFLT1-i13NM_001159920.1 2343 ACAUUAAAGGACUCAUUAAA GUAAAACAUUAAAGGACUCAUUAAAAAGUAsFLT1-i13 NM_001159920.1 2351 GGACUCAUUAAAAAGUAACAUUAAAGGACUCAUUAAAAAGUAACAGUUGU sFLT1-i13 NM_001159920.1 2353ACUCAUUAAAAAGUAACAGU AAAGGACUCAUUAAAAAGUAACAGUUGUCU sFLT1-i13NM_001159920.1 2362 AAAGUAACAGUUGUCUCAUA AUUAAAAAGUAACAGUUGUCUCAUAUCAUCsFLT1-i15a NM_001160030.1 2471 CAUCAUCAUCAUCAUAGCUAGUCAUCAUCAUCAUCAUCAUAGCUAUCAUC sFLT1-i15a NM_001160030.1 2474CAUCAUCAUCAUAGCUAUCA AUCAUCAUCAUCAUCAUAGCUAUCAUCAUU sFLT1-i15aNM_001160030.1 2477 CAUCAUCAUAGCUAUCAUCA AUCAUCAUCAUCAUAGCUAUCAUCAUUAUCsFLT1-i15a NM_001160030.1 2508 AUCAUCAUCAUCAUCAUAGCUCAUCAUCAUCAUCAUCAUCAUAGCUACCA sFLT1-i15a NM_001160030.1 2510CAUCAUCAUCAUCAUAGCUA AUCAUCAUCAUCAUCAUCAUAGCUACCAUU sFLT1-i15aNM_001160030.1 2513 CAUCAUCAUCAUAGCUACCA AUCAUCAUCAUCAUCAUAGCUACCAUUUAUsFLT1-i15a NM_001160030.1 2518 UCAUCAUAGCUACCAUUUAUCAUCAUCAUCAUAGCUACCAUUUAUUGAAA sFLT1-i15a NM_001160030.1 2519CAUCAUAGCUACCAUUUAUU AUCAUCAUCAUAGCUACCAUUUAUUGAAAA sFLT1-i15aNM_001160030.1 2525 AGCUACCAUUUAUUGAAAAC AUCAUAGCUACCAUUUAUUGAAAACUAUUAsFLT1-i15a NM_001160030.1 2528 UACCAUUUAUUGAAAACUAUAUAGCUACCAUUUAUUGAAAACUAUUAUGU sFLT1-i15a NM_001160030.1 2556AACUUCAAAGAACUUAUCCU GUGUCAACUUCAAAGAACUUAUCCUUUAGU sFLT1-i15aNM_001160030.1 2561 CAAAGAACUUAUCCUUUAGU AACUUCAAAGAACUUAUCCUUUAGUUGGAGsFLT1-i15a NM_001160030.1 2572 UCCUUUAGUUGGAGAGCCAAACUUAUCCUUUAGUUGGAGAGCCAAGACAA sFLT1-i15a NM_001160030.1 2574CUUUAGUUGGAGAGCCAAGA UUAUCCUUUAGUUGGAGAGCCAAGACAAUC sFLT1-i15aNM_001160030.1 2576 UUAGUUGGAGAGCCAAGACA AUCCUUUAGUUGGAGAGCCAAGACAAUCAUsFLT1-i15a NM_001160030.1 2577 UAGUUGGAGAGCCAAGACAAUCCUUUAGUUGGAGAGCCAAGACAAUCAUA sFLT1-i15a NM_001160030.1 2580UUGGAGAGCCAAGACAAUCA UUUAGUUGGAGAGCCAAGACAAUCAUAACA sFLT1-i15aNM_001160030.1 2582 GGAGAGCCAAGACAAUCAUA UAGUUGGAGAGCCAAGACAAUCAUAACAAUsFLT1-i15a NM_001160030.1 2585 GAGCCAAGACAAUCAUAACAUUGGAGAGCCAAGACAAUCAUAACAAUAAC sFLT1-i15a NM_001160030.1 2588CCAAGACAAUCAUAACAAUA GAGAGCCAAGACAAUCAUAACAAUAACAAA sFLT1-i15aNM_001160030.1 2590 AAGACAAUCAUAACAAUAAC GAGCCAAGACAAUCAUAACAAUAACAAAUG

Various aspects of the invention are described in further detail in thefollowing subsections.

I. siRNA Design

In some embodiments, siRNAs are designed as follows. First, a portion ofthe target gene (e.g., the flt1 gene), e.g., one or more of the targetsequences set forth at FIG. 6, is selected, e.g., one or any combinationof sFLT1-i13-2283, sFlt1-i15a-2519, sFLT1-i13-2318, sFlt1-i15a-2585 froman intronic region of a target gene. Cleavage of mRNA at these sitesshould eliminate translation of corresponding soluble protein. Sensestrands were designed based on the target sequence. (See FIG. 13.)Preferably, the portion (and corresponding sense strand) includes about30 to 35 nucleotides, e.g., 30, 31, 32, 33, 34 or 35 nucleotides. Morepreferably, the portion (and corresponding sense strand) includes 21, 22or 23 nucleotides. The skilled artisan will appreciate, however, thatsiRNAs having a length of less than 19 nucleotides or greater than 25nucleotides can also function to mediate RNAi. Accordingly, siRNAs ofsuch length are also within the scope of the instant invention providedthat they retain the ability to mediate RNAi. Longer RNAi agents havebeen demonstrated to elicit an interferon or PKR response in certainmammalian cells which may be undesirable. Preferably, the RNAi agents ofthe invention do not elicit a PKR response (i.e., are of a sufficientlyshort length). However, longer RNAi agents may be useful, for example,in cell types incapable of generating a PRK response or in situationswhere the PKR response has been down-regulated or dampened byalternative means.

The sense strand sequence is designed such that the target sequence isessentially in the middle of the strand. Moving the target sequence toan off-center position may, in some instances, reduce efficiency ofcleavage by the siRNA. Such compositions, i.e., less efficientcompositions, may be desirable for use if off-silencing of the wild-typemRNA is detected.

The antisense strand is routinely the same length as the sense strandand includes complementary nucleotides. In one embodiment, the strandsare fully complementary, i.e., the strands are blunt-ended when alignedor annealed. In another embodiment, the strands comprise align or annealsuch that 1-, 2- or 3-nucleotide overhangs are generated, i.e., the 3′end of the sense strand extends 1, 2 or 3 nucleotides further than the5′ end of the antisense strand and/or the 3′ end of the antisense strandextends 1, 2 or 3 nucleotides further than the 5′ end of the sensestrand. Overhangs can comprise (or consist of) nucleotides correspondingto the target gene sequence (or complement thereof). Alternatively,overhangs can comprise (or consist of) deoxyribonucleotides, for exampledTs, or nucleotide analogs, or other suitable non-nucleotide material.

To facilitate entry of the antisense strand into RISC (and thus increaseor improve the efficiency of target cleavage and silencing), the basepair strength between the 5′ end of the sense strand and 3′ end of theantisense strand can be altered, e.g., lessened or reduced, as describedin detail in U.S. Pat. Nos. 7,459,547, 7,772,203 and 7,732,593, entitled“Methods and Compositions for Controlling Efficacy of RNA Silencing”(filed Jun. 2, 2003) and U.S. Pat. Nos. 8,309,704, 7,750,144, 8,304,530,8,329,892 and 8,309,705, entitled “Methods and Compositions forEnhancing the Efficacy and Specificity of RNAi” (filed Jun. 2, 2003),the contents of which are incorporated in their entirety by thisreference. In one embodiment of these aspects of the invention, thebase-pair strength is less due to fewer G:C base pairs between the 5′end of the first or antisense strand and the 3′ end of the second orsense strand than between the 3′ end of the first or antisense strandand the 5′ end of the second or sense strand. In another embodiment, thebase pair strength is less due to at least one mismatched base pairbetween the 5′ end of the first or antisense strand and the 3′ end ofthe second or sense strand. In certain exemplary embodiments, themismatched base pair is selected from the group consisting of G:A, C:A,C:U, G:G, A:A, C:C and U:U. In another embodiment, the base pairstrength is less due to at least one wobble base pair, e.g., G:U,between the 5′ end of the first or antisense strand and the 3′ end ofthe second or sense strand. In another embodiment, the base pairstrength is less due to at least one base pair comprising a rarenucleotide, e.g., inosine (I). In certain exemplary embodiments, thebase pair is selected from the group consisting of an I:A, I:U and I:C.In yet another embodiment, the base pair strength is less due to atleast one base pair comprising a modified nucleotide. In certainexemplary embodiments, the modified nucleotide is selected from thegroup consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and2,6-diamino-A.

The design of siRNAs suitable for targeting the sFlt1 target sequencesset forth at FIG. 6 is described in detail below. siRNAs can be designedaccording to the above exemplary teachings for any other targetsequences found in the flt1 gene. Moreover, the technology is applicableto targeting any other target sequences, e.g., non-disease causingtarget sequences.

To validate the effectiveness by which siRNAs destroy mRNAs (e.g., sFLT1mRNA), the siRNA can be incubated with cDNA (e.g., Flt1 cDNA) in aDrosophila-based in vitro mRNA expression system. Radiolabeled with ³²P,newly synthesized mRNAs (e.g., Flt11mRNA) are detectedautoradiographically on an agarose gel. The presence of cleaved mRNAindicates mRNA nuclease activity. Suitable controls include omission ofsiRNA. Alternatively, control siRNAs are selected having the samenucleotide composition as the selected siRNA, but without significantsequence complementarity to the appropriate target gene. Such negativecontrols can be designed by randomly scrambling the nucleotide sequenceof the selected siRNA; a homology search can be performed to ensure thatthe negative control lacks homology to any other gene in the appropriategenome. In addition, negative control siRNAs can be designed byintroducing one or more base mismatches into the sequence.

Sites of siRNA-mRNA complementation are selected which result in optimalmRNA specificity and maximal mRNA cleavage.

II. RNAi Agents

The present invention includes siRNA molecules designed, for example, asdescribed above. The siRNA molecules of the invention can be chemicallysynthesized, or can be transcribed in vitro from a DNA template, or invivo from e.g., shRNA, or by using recombinant human DICER enzyme, tocleave in vitro transcribed dsRNA templates into pools of 20-, 21- or23-bp duplex RNA mediating RNAi. The siRNA molecules can be designedusing any method known in the art.

In one aspect, instead of the RNAi agent being an interferingribonucleic acid, e.g., an siRNA or shRNA as described above, the RNAiagent can encode an interfering ribonucleic acid, e.g., an shRNA, asdescribed above. In other words, the RNAi agent can be a transcriptionaltemplate of the interfering ribonucleic acid. Thus, RNAi agents of thepresent invention can also include small hairpin RNAs (shRNAs), andexpression constructs engineered to express shRNAs. Transcription ofshRNAs is initiated at a polymerase III (pol III) promoter, and isthought to be terminated at position 2 of a 4-5-thymine transcriptiontermination site. Upon expression, shRNAs are thought to fold into astem-loop structure with 3′ UU-overhangs; subsequently, the ends ofthese shRNAs are processed, converting the shRNAs into siRNA-likemolecules of about 21-23 nucleotides (Brummelkamp et al., 2002; Lee etal., 2002, Supra; Miyagishi et al., 2002; Paddison et al., 2002, supra;Paul et al., 2002, supra; Sui et al., 2002 supra; Yu et al., 2002,supra. More information about shRNA design and use can be found on theinternet at the following addresses:katandin.cshl.org:9331/RNAi/docs/BseRI-BamHI_Strategy.pdf andkatandin.cshl.org: 9331/RNAi/docs/Web_version_of_PCR_strategy 1.pdf).

Expression constructs of the present invention include any constructsuitable for use in the appropriate expression system and include, butare not limited to, retroviral vectors, linear expression cassettes,plasmids and viral or virally-derived vectors, as known in the art. Suchexpression constructs can include one or more inducible promoters, RNAPol III promoter systems such as U6 snRNA promoters or H1 RNA polymeraseIII promoters, or other promoters known in the art. The constructs caninclude one or both strands of the siRNA. Expression constructsexpressing both strands can also include loop structures linking bothstrands, or each strand can be separately transcribed from separatepromoters within the same construct. Each strand can also be transcribedfrom a separate expression construct. (Tuschl, T., 2002, Supra).

Synthetic siRNAs can be delivered into cells by methods known in theart, including cationic liposome transfection and electroporation. Toobtain longer term suppression of the target genes (i.e., flt1 genes)and to facilitate delivery under certain circumstances, one or moresiRNA can be expressed within cells from recombinant DNA constructs.Such methods for expressing siRNA duplexes within cells from recombinantDNA constructs to allow longer-term target gene suppression in cells areknown in the art, including mammalian Pol III promoter systems (e.g., H1or U6/snRNA promoter systems (Tuschl, T., 2002, supra) capable ofexpressing functional double-stranded siRNAs; (Bagella et al., 1998; Leeet al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002,supra; Yu et al., 2002), supra; Sui et al., 2002, supra).Transcriptional termination by RNA Pol III occurs at runs of fourconsecutive T residues in the DNA template, providing a mechanism to endthe siRNA transcript at a specific sequence. The siRNA is complementaryto the sequence of the target gene in 5′-3′ and 3′-5′ orientations, andthe two strands of the siRNA can be expressed in the same construct orin separate constructs. Hairpin siRNAs, driven by H1 or U6 snRNApromoter and expressed in cells, can inhibit target gene expression(Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002,supra; Paul et al., 2002, supra; Yu et al., 2002), supra; Sui et al.,2002, supra). Constructs containing siRNA sequence under the control ofT7 promoter also make functional siRNAs when cotransfected into thecells with a vector expressing T7 RNA polymerase (Jacque et al., 2002,supra). A single construct may contain multiple sequences coding forsiRNAs, such as multiple regions of the gene encoding sFlt1, targetingthe same gene or multiple genes, and can be driven, for example, byseparate PolIII promoter sites.

Animal cells express a range of noncoding RNAs of approximately 22nucleotides termed micro RNA (miRNAs) which can regulate gene expressionat the post transcriptional or translational level during animaldevelopment. One common feature of miRNAs is that they are all excisedfrom an approximately 70 nucleotide precursor RNA stem-loop, probably byDicer, an RNase III-type enzyme, or a homolog thereof. By substitutingthe stem sequences of the miRNA precursor with sequence complementary tothe target mRNA, a vector construct that expresses the engineeredprecursor can be used to produce siRNAs to initiate RNAi againstspecific mRNA targets in mammalian cells (Zeng et al., 2002, supra).When expressed by DNA vectors containing polymerase III promoters,micro-RNA designed hairpins can silence gene expression (McManus et al.,2002, supra). MicroRNAs targeting polymorphisms may also be useful forblocking translation of mutant proteins, in the absence ofsiRNA-mediated gene-silencing. Such applications may be useful insituations, for example, where a designed siRNA caused off-targetsilencing of wild type protein.

Viral-mediated delivery mechanisms can also be used to induce specificsilencing of targeted genes through expression of siRNA, for example, bygenerating recombinant adenoviruses harboring siRNA under RNA Pol IIpromoter transcription control (Xia et al., 2002, supra). Infection ofHeLa cells by these recombinant adenoviruses allows for diminishedendogenous target gene expression. Injection of the recombinantadenovirus vectors into transgenic mice expressing the target genes ofthe siRNA results in in vivo reduction of target gene expression. Id. Inan animal model, whole-embryo electroporation can efficiently deliversynthetic siRNA into post-implantation mouse embryos (Calegari et al.,2002). In adult mice, efficient delivery of siRNA can be accomplished by“high-pressure” delivery technique, a rapid injection (within 5 seconds)of a large volume of siRNA containing solution into animal via the tailvein (Liu et al., 1999, supra; McCaffrey et al., 2002, supra; Lewis etal., 2002. Nanoparticles and liposomes can also be used to deliver siRNAinto animals. In certain exemplary embodiments, recombinantadeno-associated viruses (rAAVs) and their associated vectors can beused to deliver one or more siRNAs into cells, e.g., neural cells (e.g.,brain cells) (US Patent Applications 2014/0296486, 2010/0186103,2008/0269149, 2006/0078542 and 2005/0220766).

The nucleic acid compositions of the invention include both unmodifiedsiRNAs and modified siRNAs as known in the art, such as crosslinkedsiRNA derivatives or derivatives having non nucleotide moieties linked,for example to their 3′ or 5′ ends. Modifying siRNA derivatives in thisway may improve cellular uptake or enhance cellular targeting activitiesof the resulting siRNA derivative as compared to the correspondingsiRNA, are useful for tracing the siRNA derivative in the cell, orimprove the stability of the siRNA derivative compared to thecorresponding siRNA.

Engineered RNA precursors, introduced into cells or whole organisms asdescribed herein, will lead to the production of a desired siRNAmolecule. Such an siRNA molecule will then associate with endogenousprotein components of the RNAi pathway to bind to and target a specificmRNA sequence for cleavage and destruction. In this fashion, the mRNA tobe targeted by the siRNA generated from the engineered RNA precursorwill be depleted from the cell or organism, leading to a decrease in theconcentration of the protein encoded by that mRNA in the cell ororganism. The RNA precursors are typically nucleic acid molecules thatindividually encode either one strand of a dsRNA or encode the entirenucleotide sequence of an RNA hairpin loop structure.

The nucleic acid compositions of the invention can be unconjugated orcan be conjugated to another moiety, such as a nanoparticle, to enhancea property of the compositions, e.g., a pharmacokinetic parameter suchas absorption, efficacy, bioavailability and/or half-life. Theconjugation can be accomplished by methods known in the art, e.g., usingthe methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001)(describes nucleic acids loaded to polyalkylcyanoacrylate (PACA)nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998)(describes nucleic acids bound to nanoparticles); Schwab et al., Ann.Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked tointercalating agents, hydrophobic groups, polycations or PACAnanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995)(describes nucleic acids linked to nanoparticles).

The nucleic acid molecules of the present invention can also be labeledusing any method known in the art. For instance, the nucleic acidcompositions can be labeled with a fluorophore, e.g., Cy3, fluorescein,or rhodamine. The labeling can be carried out using a kit, e.g., theSILENCER™ siRNA labeling kit (Ambion). Additionally, the siRNA can beradiolabeled, e.g., using ³H, ³²P or other appropriate isotope.

Moreover, because RNAi is believed to progress via at least onesingle-stranded RNA intermediate, the skilled artisan will appreciatethat ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also bedesigned (e.g., for chemical synthesis) generated (e.g., enzymaticallygenerated) or expressed (e.g., from a vector or plasmid) as describedherein and utilized according to the claimed methodologies. Moreover, ininvertebrates, RNAi can be triggered effectively by long dsRNAs (e.g.,dsRNAs about 100-1000 nucleotides in length, preferably about 200-500,for example, about 250, 300, 350, 400 or 450 nucleotides in length)acting as effectors of RNAi. (Brondani et al., Proc Natl Acad Sci USA.2001 Dec. 4; 98(25):14428-33. Epub 2001 Nov. 27.)

III. Anti-sFlt1 RNA Silencing Agents

The present invention features anti-sFlt1 RNA silencing agents (e.g.,siRNA and shRNAs), methods of making said RNA silencing agents, andmethods (e.g., research and/or therapeutic methods) for using saidimproved RNA silencing agents (or portions thereof) for RNA silencing ofone or more sFLT-1 proteins. The RNA silencing agents comprise anantisense strand (or portions thereof), wherein the antisense strand hassufficient complementary to a heterozygous single nucleotidepolymorphism to mediate an RNA-mediated silencing mechanism (e.g. RNAi).

a) Design of Anti-sFlt1 siRNA Molecules

An siRNA molecule of the invention is a duplex consisting of a sensestrand and complementary antisense strand, the antisense strand havingsufficient complementary to an sFlt1 mRNA to mediate RNAi. Preferably,the siRNA molecule has a length from about 10-50 or more nucleotides,i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs).More preferably, the siRNA molecule has a length from about 16-30, e.g.,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides in each strand, wherein one of the strands is sufficientlycomplementary to a target region. Preferably, the strands are alignedsuch that there are at least 1, 2, or 3 bases at the end of the strandswhich do not align (i.e., for which no complementary bases occur in theopposing strand) such that an overhang of 1, 2 or 3 residues occurs atone or both ends of the duplex when strands are annealed. Preferably,the siRNA molecule has a length from about 10-50 or more nucleotides,i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs).More preferably, the siRNA molecule has a length from about 16-30, e.g.,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides in each strand, wherein one of the strands is substantiallycomplementary to a target sequence, and the other strand is identical orsubstantially identical to the first strand.

Generally, siRNAs can be designed by using any method known in the art,for instance, by using the following protocol:

1. The siRNA should be specific for a target sequence, e.g., a targetsequence set forth in FIG. 6 or 13. In one embodiment, a target sequenceis found in a soluble Flt1 mRNA, but not in the full-length Flt mRNA. Inanother embodiment, a target sequence is found in both a soluble Flt1mRNA and the full-length Flt mRNA. In another embodiment, a targetsequence is found in the full-length Flt mRNA. The first strand shouldbe complementary to the target sequence, and the other strand issubstantially complementary to the first strand. (See FIG. 6 forexemplary sense and antisense strands.) In one embodiment, the targetsequence is encoded in an intronic region of one or more soluble FltmRNA sequences. Exemplary target sequences correspond to one or moreintronic regions of a target gene. Cleavage of mRNA at these sitesshould eliminate translation of corresponding soluble protein but not ofthe full-length protein. Target sequences from other regions of the fitgene are also suitable for targeting. A sense strand is designed basedon the target sequence. Further, siRNAs with lower G/C content (35-55%)may be more active than those with G/C content higher than 55%. Thus inone embodiment, the invention includes nucleic acid molecules having35-55% G/C content.

2. The sense strand of the siRNA is designed based on the sequence ofthe selected target site. Preferably the sense strand includes about 19to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25 nucleotides. Morepreferably, the sense strand includes 21, 22 or 23 nucleotides. Theskilled artisan will appreciate, however, that siRNAs having a length ofless than 19 nucleotides or greater than 25 nucleotides can alsofunction to mediate RNAi. Accordingly, siRNAs of such length are alsowithin the scope of the instant invention provided that they retain theability to mediate RNAi. Longer RNA silencing agents have beendemonstrated to elicit an interferon or Protein Kinase R (PKR) responsein certain mammalian cells which may be undesirable. Preferably the RNAsilencing agents of the invention do not elicit a PKR response (i.e.,are of a sufficiently short length). However, longer RNA silencingagents may be useful, for example, in cell types incapable of generatinga PRK response or in situations where the PKR response has beendown-regulated or dampened by alternative means.

The siRNA molecules of the invention have sufficient complementaritywith the target sequence such that the siRNA can mediate RNAi. Ingeneral, siRNA containing nucleotide sequences sufficiently identical toa target sequence portion of the target gene to effect RISC-mediatedcleavage of the target gene are preferred. Accordingly, in a preferredembodiment, the sense strand of the siRNA is designed have to have asequence sufficiently identical to a portion of the target. For example,the sense strand may have 100% identity to the target site. However,100% identity is not required. Greater than 80% identity, e.g., 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or even 100% identity, between the sense strandand the target RNA sequence is preferred. The invention has theadvantage of being able to tolerate certain sequence variations toenhance efficiency and specificity of RNAi. In one embodiment, the sensestrand has 4, 3, 2, 1, or 0 mismatched nucleotide(s) with a targetregion, such as a target region that differs by at least one base pairbetween a soluble flt1 and a full-length flt1 allele, e.g., a targetregion comprising the gain-of-function mutation, and the other strand isidentical or substantially identical to the first strand. Moreover,siRNA sequences with small insertions or deletions of 1 or 2 nucleotidesmay also be effective for mediating RNAi. Alternatively, siRNA sequenceswith nucleotide analog substitutions or insertions can be effective forinhibition.

Sequence identity may be determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=number of identical positions/totalnumber of positions×100), optionally penalizing the score for the numberof gaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

3. The antisense or guide strand of the siRNA is routinely the samelength as the sense strand and includes complementary nucleotides. Inone embodiment, the guide and sense strands are fully complementary,i.e., the strands are blunt-ended when aligned or annealed. In anotherembodiment, the strands of the siRNA can be paired in such a way as tohave a 3′ overhang of 1 to 4, e.g., 2, nucleotides. Overhangs cancomprise (or consist of) nucleotides corresponding to the target genesequence (or complement thereof). Alternatively, overhangs can comprise(or consist of) deoxyribonucleotides, for example dTs, or nucleotideanalogs, or other suitable non-nucleotide material. Thus in anotherembodiment, the nucleic acid molecules may have a 3′ overhang of 2nucleotides, such as TT. The overhanging nucleotides may be either RNAor DNA. As noted above, it is desirable to choose a target regionwherein the mutant:wild type mismatch is a purine:purine mismatch.

4. Using any method known in the art, compare the potential targets tothe appropriate genome database (human, mouse, rat, etc.) and eliminatefrom consideration any target sequences with significant homology toother coding sequences. One such method for such sequence homologysearches is known as BLAST, which is available at National Center forBiotechnology Information website.

5. Select one or more sequences that meet your criteria for evaluation.

Further general information about the design and use of siRNA may befound in “The siRNA User Guide,” available at The Max-Plank-Institut furBiophysikalishe Chemie website.

Alternatively, the siRNA may be defined functionally as a nucleotidesequence (or oligonucleotide sequence) that is capable of hybridizingwith the target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing). Additional preferred hybridization conditions includehybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C. in 0.3×SSC or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The hybridization temperature for hybrids anticipated to be lessthan 50 base pairs in length should be 5-10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] isthe concentration of sodium ions in the hybridization buffer ([Na+] for1×SSC=0.165 M). Additional examples of stringency conditions forpolynucleotide hybridization are provided in Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11, and Current Protocols in Molecular Biology, 1995, F. M.Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4, incorporated herein by reference.

Negative control siRNAs should have the same nucleotide composition asthe selected siRNA, but without significant sequence complementarity tothe appropriate genome. Such negative controls may be designed byrandomly scrambling the nucleotide sequence of the selected siRNA. Ahomology search can be performed to ensure that the negative controllacks homology to any other gene in the appropriate genome. In addition,negative control siRNAs can be designed by introducing one or more basemismatches into the sequence.

6. To validate the effectiveness by which siRNAs destroy target mRNAs(e.g., sFlt1 mRNA corresponding to soluble FLT1), the siRNA may beincubated with target cDNA (e.g., flt1 cDNA) in a Drosophila-based invitro mRNA expression system. Radiolabeled with ³²P, newly synthesizedtarget mRNAs (e.g., sFlt1 mRNA) are detected autoradiographically on anagarose gel. The presence of cleaved target mRNA indicates mRNA nucleaseactivity. Suitable controls include omission of siRNA and use ofnon-target cDNA. Alternatively, control siRNAs are selected having thesame nucleotide composition as the selected siRNA, but withoutsignificant sequence complementarity to the appropriate target gene.Such negative controls can be designed by randomly scrambling thenucleotide sequence of the selected siRNA. A homology search can beperformed to ensure that the negative control lacks homology to anyother gene in the appropriate genome. In addition, negative controlsiRNAs can be designed by introducing one or more base mismatches intothe sequence.

Anti-sflt1 siRNAs may be designed to target any of the target sequencesdescribed supra. Said siRNAs comprise an antisense strand which issufficiently complementary with the target sequence to mediate silencingof the target sequence. In certain embodiments, the RNA silencing agentis a siRNA.

In certain embodiments, the siRNA comprises a sense strand comprising asequence set forth at FIG. 6, and an antisense strand comprising asequence set forth at FIG. 6.

Sites of siRNA-mRNA complementation are selected which result in optimalmRNA specificity and maximal mRNA cleavage.

b) siRNA-Like Molecules

siRNA-like molecules of the invention have a sequence (i.e., have astrand having a sequence) that is “sufficiently complementary” to atarget sequence of a sflt1 mRNA to direct gene silencing either by RNAior translational repression. siRNA-like molecules are designed in thesame way as siRNA molecules, but the degree of sequence identity betweenthe sense strand and target RNA approximates that observed between anmiRNA and its target. In general, as the degree of sequence identitybetween a miRNA sequence and the corresponding target gene sequence isdecreased, the tendency to mediate post-transcriptional gene silencingby translational repression rather than RNAi is increased. Therefore, inan alternative embodiment, where post-transcriptional gene silencing bytranslational repression of the target gene is desired, the miRNAsequence has partial complementarity with the target gene sequence. Incertain embodiments, the miRNA sequence has partial complementarity withone or more short sequences (complementarity sites) dispersed within thetarget mRNA (e.g. within the 3′-UTR of the target mRNA) (Hutvagner andZamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA,2003; Doench et al., Genes & Dev., 2003). Since the mechanism oftranslational repression is cooperative, multiple complementarity sites(e.g., 2, 3, 4, 5 or 6) may be targeted in certain embodiments.

The capacity of a siRNA-like duplex to mediate RNAi or translationalrepression may be predicted by the distribution of non-identicalnucleotides between the target gene sequence and the nucleotide sequenceof the silencing agent at the site of complementarity. In oneembodiment, where gene silencing by translational repression is desired,at least one non-identical nucleotide is present in the central portionof the complementarity site so that duplex formed by the miRNA guidestrand and the target mRNA contains a central “bulge” (Doench J G etal., Genes & Dev., 2003). In another embodiment 2, 3, 4, 5 or 6contiguous or non-contiguous non-identical nucleotides are introduced.The non-identical nucleotide may be selected such that it forms a wobblebase pair (e.g., G:U) or a mismatched base pair (G:A, C:A, C:U, G:G,A:A, C:C, U:U). In a further preferred embodiment, the “bulge” iscentered at nucleotide positions 12 and 13 from the 5′ end of the miRNAmolecule.

c) Short Hairpin RNA (shRNA) Molecules

In certain featured embodiments, the instant invention provides shRNAscapable of mediating RNA silencing of an sFlt1 target sequence withenhanced selectivity. In contrast to siRNAs, shRNAs mimic the naturalprecursors of micro RNAs (miRNAs) and enter at the top of the genesilencing pathway. For this reason, shRNAs are believed to mediate genesilencing more efficiently by being fed through the entire natural genesilencing pathway.

miRNAs are noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel during plant and animal development. One common feature of miRNAsis that they are all excised from an approximately 70 nucleotideprecursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNaseIII-type enzyme, or a homolog thereof. Naturally-occurring miRNAprecursors (pre-miRNA) have a single strand that forms a duplex stemincluding two portions that are generally complementary, and a loop,that connects the two portions of the stem. In typical pre-miRNAs, thestem includes one or more bulges, e.g., extra nucleotides that create asingle nucleotide “loop” in one portion of the stem, and/or one or moreunpaired nucleotides that create a gap in the hybridization of the twoportions of the stem to each other. Short hairpin RNAs, or engineeredRNA precursors, of the invention are artificial constructs based onthese naturally occurring pre-miRNAs, but which are engineered todeliver desired RNA silencing agents (e.g., siRNAs of the invention). Bysubstituting the stem sequences of the pre-miRNA with sequencecomplementary to the target mRNA, a shRNA is formed. The shRNA isprocessed by the entire gene silencing pathway of the cell, therebyefficiently mediating RNAi.

The requisite elements of a shRNA molecule include a first portion and asecond portion, having sufficient complementarity to anneal or hybridizeto form a duplex or double-stranded stem portion. The two portions neednot be fully or perfectly complementary. The first and second “stem”portions are connected by a portion having a sequence that hasinsufficient sequence complementarity to anneal or hybridize to otherportions of the shRNA. This latter portion is referred to as a “loop”portion in the shRNA molecule. The shRNA molecules are processed togenerate siRNAs. shRNAs can also include one or more bulges, i.e., extranucleotides that create a small nucleotide “loop” in a portion of thestem, for example a one-, two- or three-nucleotide loop. The stemportions can be the same length, or one portion can include an overhangof, for example, 1-5 nucleotides. The overhanging nucleotides caninclude, for example, uracils (Us), e.g., all Us. Such Us are notablyencoded by thymidines (Ts) in the shRNA-encoding DNA which signal thetermination of transcription.

In shRNAs (or engineered precursor RNAs) of the instant invention, oneportion of the duplex stem is a nucleic acid sequence that iscomplementary (or antisense) to the sFlt1 target sequence. Preferably,one strand of the stem portion of the shRNA is sufficientlycomplementary (e.g., antisense) to a target RNA (e.g., mRNA) sequence tomediate degradation or cleavage of said target RNA via RNA interference(RNAi). Thus, engineered RNA precursors include a duplex stem with twoportions and a loop connecting the two stem portions. The antisenseportion can be on the 5′ or 3′ end of the stem. The stem portions of ashRNA are preferably about 15 to about 50 nucleotides in length.Preferably the two stem portions are about 18 or 19 to about 21, 22, 23,24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. Inpreferred embodiments, the length of the stem portions should be 21nucleotides or greater. When used in mammalian cells, the length of thestem portions should be less than about 30 nucleotides to avoidprovoking non-specific responses like the interferon pathway. Innon-mammalian cells, the stem can be longer than 30 nucleotides. Infact, the stem can include much larger sections complementary to thetarget mRNA (up to, and including the entire mRNA). In fact, a stemportion can include much larger sections complementary to the targetmRNA (up to, and including the entire mRNA).

The two portions of the duplex stem must be sufficiently complementaryto hybridize to form the duplex stem. Thus, the two portions can be, butneed not be, fully or perfectly complementary. In addition, the two stemportions can be the same length, or one portion can include an overhangof 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include,for example, uracils (Us), e.g., all Us. The loop in the shRNAs orengineered RNA precursors may differ from natural pre-miRNA sequences bymodifying the loop sequence to increase or decrease the number of pairednucleotides, or replacing all or part of the loop sequence with atetraloop or other loop sequences. Thus, the loop in the shRNAs orengineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g.,15 or 20, or more nucleotides in length.

The loop in the shRNAs or engineered RNA precursors may differ fromnatural pre-miRNA sequences by modifying the loop sequence to increaseor decrease the number of paired nucleotides, or replacing all or partof the loop sequence with a tetraloop or other loop sequences. Thus, theloop portion in the shRNA can be about 2 to about 20 nucleotides inlength, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, ormore nucleotides in length. A preferred loop consists of or comprises a“tetraloop” sequences. Exemplary tetraloop sequences include, but arenot limited to, the sequences GNRA, where N is any nucleotide and R is apurine nucleotide, GGGG, and UUUU.

In certain embodiments, shRNAs of the invention include the sequences ofa desired siRNA molecule described supra. In other embodiments, thesequence of the antisense portion of a shRNA can be designed essentiallyas described above or generally by selecting an 18, 19, 20, 21nucleotide, or longer, sequence from within the target RNA (e.g., sflt1mRNA), for example, from a region 100 to 200 or 300 nucleotides upstreamor downstream of the start of translation. In general, the sequence canbe selected from any portion of the target RNA (e.g., mRNA) including anintronic region, the 5′ UTR (untranslated region), coding sequence, or3′ UTR, provided said portion is distant from the site of thegain-of-function mutation. This sequence can optionally followimmediately after a region of the target gene containing two adjacent AAnucleotides. The last two nucleotides of the nucleotide sequence can beselected to be UU. This 21 or so nucleotide sequence is used to createone portion of a duplex stem in the shRNA. This sequence can replace astem portion of a wild-type pre-miRNA sequence, e.g., enzymatically, oris included in a complete sequence that is synthesized. For example, onecan synthesize DNA oligonucleotides that encode the entire stem-loopengineered RNA precursor, or that encode just the portion to be insertedinto the duplex stem of the precursor, and using restriction enzymes tobuild the engineered RNA precursor construct, e.g., from a wild-typepre-miRNA.

Engineered RNA precursors include in the duplex stem the 21-22 or sonucleotide sequences of the siRNA or siRNA-like duplex desired to beproduced in vivo. Thus, the stem portion of the engineered RNA precursorincludes at least 18 or 19 nucleotide pairs corresponding to thesequence of an exonic portion of the gene whose expression is to bereduced or inhibited. The two 3′ nucleotides flanking this region of thestem are chosen so as to maximize the production of the siRNA from theengineered RNA precursor and to maximize the efficacy of the resultingsiRNA in targeting the corresponding mRNA for translational repressionor destruction by RNAi in vivo and in vitro.

In certain embodiments, shRNAs of the invention include miRNA sequences,optionally end-modified miRNA sequences, to enhance entry into RISC. ThemiRNA sequence can be similar or identical to that of any naturallyoccurring miRNA (see e.g. The miRNA Registry; Griffiths-Jones S, Nuc.Acids Res., 2004). Over one thousand natural miRNAs have been identifiedto date and together they are thought to comprise about 1% of allpredicted genes in the genome. Many natural miRNAs are clusteredtogether in the introns of pre-mRNAs and can be identified in silicousing homology-based searches (Pasquinelli et al., 2000; Lagos-Quintanaet al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computeralgorithms (e.g. MiRScan, MiRSeeker) that predict the capability of acandidate miRNA gene to form the stem loop structure of a pri-mRNA (Gradet al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al.,Science, 2003; Lai E C et al., Genome Bio., 2003). An online registryprovides a searchable database of all published miRNA sequences (ThemiRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc.Acids Res., 2004). Exemplary, natural miRNAs include lin-4, let-7,miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs,as well as other natural miRNAs from humans and certain model organismsincluding Drosophila melanogaster, Caenorhabditis elegans, zebrafish,Arabidopsis thalania, Mus musculus, and Rattus norvegicus as describedin International PCT Publication No. WO 03/029459.

Naturally-occurring miRNAs are expressed by endogenous genes in vivo andare processed from a hairpin or stem-loop precursor (pre-miRNA orpri-miRNAs) by Dicer or other RNAses (Lagos-Quintana et al., Science,2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001;Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev.,2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003;Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al.,Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can existtransiently in vivo as a double-stranded duplex, but only one strand istaken up by the RISC complex to direct gene silencing. Certain miRNAs,e.g., plant miRNAs, have perfect or near-perfect complementarity totheir target mRNAs and, hence, direct cleavage of the target mRNAs.Other miRNAs have less than perfect complementarity to their targetmRNAs and, hence, direct translational repression of the target mRNAs.The degree of complementarity between an miRNA and its target mRNA isbelieved to determine its mechanism of action. For example, perfect ornear-perfect complementarity between a miRNA and its target mRNA ispredictive of a cleavage mechanism (Yekta et al., Science, 2004),whereas less than perfect complementarity is predictive of atranslational repression mechanism. In particular embodiments, the miRNAsequence is that of a naturally-occurring miRNA sequence, the aberrantexpression or activity of which is correlated with an miRNA disorder.

d) Dual Functional Oligonucleotide Tethers

In other embodiments, the RNA silencing agents of the present inventioninclude dual functional oligonucleotide tethers useful for theintercellular recruitment of a miRNA. Animal cells express a range ofmiRNAs, noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel. By binding a miRNA bound to RISC and recruiting it to a targetmRNA, a dual functional oligonucleotide tether can repress theexpression of genes involved e.g., in the arteriosclerotic process. Theuse of oligonucleotide tethers offer several advantages over existingtechniques to repress the expression of a particular gene. First, themethods described herein allow an endogenous molecule (often present inabundance), an miRNA, to mediate RNA silencing. Accordingly, the methodsdescribed herein obviate the need to introduce foreign molecules (e.g.,siRNAs) to mediate RNA silencing. Second, the RNA-silencing agents and,in particular, the linking moiety (e.g., oligonucleotides such as the2′-O-methyl oligonucleotide), can be made stable and resistant tonuclease activity. As a result, the tethers of the present invention canbe designed for direct delivery, obviating the need for indirectdelivery (e.g. viral) of a precursor molecule or plasmid designed tomake the desired agent within the cell. Third, tethers and theirrespective moieties, can be designed to conform to specific mRNA sitesand specific miRNAs. The designs can be cell and gene product specific.Fourth, the methods disclosed herein leave the mRNA intact, allowing oneskilled in the art to block protein synthesis in short pulses using thecell's own machinery. As a result, these methods of RNA silencing arehighly regulatable.

The dual functional oligonucleotide tethers (“tethers”) of the inventionare designed such that they recruit miRNAs (e.g., endogenous cellularmiRNAs) to a target mRNA so as to induce the modulation of a gene ofinterest. In preferred embodiments, the tethers have the formula T-L-μ,wherein T is an mRNA targeting moiety, L is a linking moiety, and t isan miRNA recruiting moiety. Any one or more moiety may be doublestranded. Preferably, however, each moiety is single stranded.

Moieties within the tethers can be arranged or linked (in the 5′ to 3′direction) as depicted in the formula T-L-μ (i.e., the 3′ end of thetargeting moiety linked to the 5′ end of the linking moiety and the 3′end of the linking moiety linked to the 5′ end of the miRNA recruitingmoiety). Alternatively, the moieties can be arranged or linked in thetether as follows: μ-T-L (i.e., the 3′ end of the miRNA recruitingmoiety linked to the 5′ end of the linking moiety and the 3′ end of thelinking moiety linked to the 5′ end of the targeting moiety).

The mRNA targeting moiety, as described above, is capable of capturing aspecific target mRNA. According to the invention, expression of thetarget mRNA is undesirable, and, thus, translational repression of themRNA is desired. The mRNA targeting moiety should be of sufficient sizeto effectively bind the target mRNA. The length of the targeting moietywill vary greatly depending, in part, on the length of the target mRNAand the degree of complementarity between the target mRNA and thetargeting moiety. In various embodiments, the targeting moiety is lessthan about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, or 5 nucleotides in length. In a particular embodiment,the targeting moiety is about 15 to about 25 nucleotides in length.

The miRNA recruiting moiety, as described above, is capable ofassociating with a miRNA. According to the invention, the miRNA may beany miRNA capable of repressing the target mRNA (e.g., one or more sflt1mRNAs). Mammals are reported to have over 250 endogenous miRNAs(Lagos-Quintana et al. (2002) Current Biol. 12:735-739; Lagos-Quintanaet al. (2001) Science 294:858-862; and Lim et al. (2003) Science299:1540). In various embodiments, the miRNA may be any art-recognizedmiRNA.

The linking moiety is any agent capable of linking the targetingmoieties such that the activity of the targeting moieties is maintained.Linking moieties are preferably oligonucleotide moieties comprising asufficient number of nucleotides such that the targeting agents cansufficiently interact with their respective targets. Linking moietieshave little or no sequence homology with cellular mRNA or miRNAsequences. Exemplary linking moieties include one or more2′-O-methylnucleotides, e.g., 2′-β-methyladenosine,2′-O-methylthymidine, 2′-O-methylguanosine or 2′-O-methyluridine.

e) Gene Silencing Oligonucleotides

In certain exemplary embodiments, gene expression (i.e., sflt1 geneexpression) can be modulated using oligonucleotide-based compoundscomprising two or more single stranded antisense oligonucleotides thatare linked through their 5′-ends that allow the presence of two or moreaccessible 3′-ends to effectively inhibit or decrease sflt1 geneexpression. Such linked oligonucleotides are also known as GeneSilencing Oligonucleotides (GSOs). (See, e.g., U.S. Pat. No. 8,431,544assigned to Idera Pharmaceuticals, Inc., incorporated herein byreference in its entirety for all purposes.)

The linkage at the 5′ ends of the GSOs is independent of the otheroligonucleotide linkages and may be directly via 5′, 3′ or 2′ hydroxylgroups, or indirectly, via a non-nucleotide linker or a nucleoside,utilizing either the 2′ or 3′ hydroxyl positions of the nucleoside.Linkages may also utilize a functionalized sugar or nucleobase of a 5′terminal nucleotide.

GSOs can comprise two identical or different sequences conjugated attheir 5′-5′ ends via a phosphodiester, phosphorothioate ornon-nucleoside linker. Such compounds may comprise 15 to 27 nucleotidesthat are complementary to specific portions of mRNA targets of interestfor antisense down regulation of gene product. GSOs that compriseidentical sequences can bind to a specific mRNA via Watson-Crickhydrogen bonding interactions and inhibit protein expression. GSOs thatcomprise different sequences are able to bind to two or more differentregions of one or more mRNA target and inhibit protein expression. Suchcompounds are comprised of heteronucleotide sequences complementary totarget mRNA and form stable duplex structures through Watson-Crickhydrogen bonding. Under certain conditions, GSOs containing two free3′-ends (5′-5′-attached antisense) can be more potent inhibitors of geneexpression than those containing a single free 3′-end or no free 3′-end.

In some embodiments, the non-nucleotide linker is glycerol or a glycerolhomolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and pindependently are integers from 1 to about 6, from 1 to about 4 or from1 to about 3. In some other embodiments, the non-nucleotide linker is aderivative of 1,3-diamino-2-hydroxypropane. Some such derivatives havethe formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH,wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 toabout 6 or from 2 to about 4.

Some non-nucleotide linkers permit attachment of more than two GSOcomponents. For example, the non-nucleotide linker glycerol has threehydroxyl groups to which GSO components may be covalently attached. Someoligonucleotide-based compounds of the invention, therefore, comprisetwo or more oligonucleotides linked to a nucleotide or a non-nucleotidelinker. Such oligonucleotides according to the invention are referred toas being “branched.”

In certain embodiments, GSOs are at least 14 nucleotides in length. Incertain exemplary embodiments, GSOs are 15 to 40 nucleotides long or 20to 30 nucleotides in length. Thus, the component oligonucleotides ofGSOs can independently be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40nucleotides in length.

These oligonucleotides can be prepared by the art recognized methodssuch as phosphoramidate or H-phosphonate chemistry which can be carriedout manually or by an automated synthesizer. These oligonucleotides mayalso be modified in a number of ways without compromising their abilityto hybridize to mRNA. Such modifications may include at least oneinternucleotide linkage of the oligonucleotide being analkylphosphonate, phosphorothioate, phosphorodithioate,methylphosphonate, phosphate ester, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate hydroxyl, acetamidateor carboxymethyl ester or a combination of these and otherinternucleotide linkages between the 5′ end of one nucleotide and the 3′end of another nucleotide in which the 5′ nucleotide phosphodiesterlinkage has been replaced with any number of chemical groups.

IV. Modified Anti-sFlt1 RNA Silencing Agents

In certain aspects of the invention, an RNA silencing agent (or anyportion thereof) of the invention as described supra may be modifiedsuch that the activity of the agent is further improved. For example,the RNA silencing agents described in herein may be modified with any ofthe modifications described infra. The modifications can, in part, serveto further enhance target discrimination, to enhance stability of theagent (e.g., to prevent degradation), to promote cellular uptake, toenhance the target efficiency, to improve efficacy in binding (e.g., tothe targets), to improve patient tolerance to the agent, and/or toreduce toxicity.

In certain embodiments, siRNA compounds are provided having one or anycombination of the following properties: (1) fully chemically-stabilized(i.e., no unmodified 2′-OH residues); (2) asymmetry; (3) 11-16 base pairduplexes; (4) alternating pattern of chemically-modified nucleotides(e.g., 2′-fluoro and 2′-methoxy modifications); and (5) single-stranded,fully phosphorothioated tails of 5-8 bases. The number ofphosphorothioate modifications is critical. This number is varied from 6to 17 total in different embodiments.

In certain embodiments, the siRNA compounds described herein can beconjugated to a variety of targeting agents, including, but not limitedto, cholesterol, DHA, phenyltropanes, cortisol, vitamin A, vitamin D,GalNac, and gangliozides. The cholesterol-modified version showed 5-10fold improvement in efficacy in vitro versus previously used chemicalstabilization patterns (e.g., wherein all purine but not purimidines aremodified) in wide range of cell types (e.g., HeLa, neurons, hepatocytes,trophoblasts).

Certain compounds of the invention having the structural propertiesdescribed above and herein may be referred to as “hsiRNA-ASP”(hydrophobically-modified, small interfering RNA, featuring an advancedstabilization pattern). In addition, this hsiRNA-ASP pattern showed adramatically improved distribution through the brain, spinal cord,delivery to liver, placenta, kidney, spleen and several other tissues,making them accessible for therapeutic intervention.

In liver hsiRNA-ASP delivery specifically to endothelial and kuppercells, but not hepatocytes, making this chemical modification patterncomplimentary rather than competitive technology to GalNac conjugates.

The compounds of the invention can be described in the following aspectsand embodiments.

In a first aspect, provided herein is oligonucleotide of at least 16contiguous nucleotides, said oligonucleotide having a 5′ end, a 3′ endand complementarity to a target, wherein: (1) the oligonucleotidecomprises alternating 2′-methoxy-ribonucleotides and2′-fluoro-ribonucleotides; (2) the nucleotides at positions 2 and 14from the 5′ end are not 2′-methoxy-ribonucleotides; (3) the nucleotidesare connected via phosphodiester or phosphorothioate linkages; and (4)the nucleotides at positions 1-6 from the 3′ end, or positions 1-7 fromthe 3′ end, are connected to adjacent nucleotides via phosphorothioatelinkages.

In a second aspect, provided herein is a double-stranded,chemically-modified nucleic acid, comprising a first oligonucleotide anda second oligonucleotide, wherein: (1) the first oligonucleotide is anoligonucleotide described herein (e.g., comprising SEQ ID Nos:1, 2, 3 or4); (2) a portion of the first oligonucleotide is complementary to aportion of the second oligonucleotide; (3) the second oligonucleotidecomprises alternating 2′-methoxy-ribonucleotides and2′-fluoro-ribonucleotides; (4) the nucleotides at positions 2 and 14from the 3′ end of the second oligonucleotide are2′-methoxy-ribonucleotides; and (5) the nucleotides of the secondoligonucleotide are connected via phosphodiester or phosphorothioatelinkages.

In a third aspect, provided herein is oligonucleotide having thestructure:X-A(-L-B-L-A)j(-S-B-S-A)r(-S-B)t-ORwherein: X is a 5′ phosphate group; A, for each occurrence,independently is a 2′-methoxy-ribonucleotide; B, for each occurrence,independently is a 2′-fluoro-ribonucleotide; L, for each occurrenceindependently is a phosphodiester or phosphorothioate linker; S is aphosphorothioate linker; and R is selected from hydrogen and a cappinggroup (e.g., an acyl such as acetyl); j is 4, 5, 6 or 7; r is 2 or 3;and t is 0 or 1.

In a fourth aspect, provided herein is a double-stranded,chemically-modified nucleic acid comprising a first oligonucleotide anda second oligonucleotide, wherein: (1) the first oligonucleotide isselected from the oligonucleotides of the third aspect; (2) a portion ofthe first oligonucleotide is complementary to a portion of the secondoligonucleotide; and (3) the second oligonucleotide has the structure:C-L-B(-S-A-S-B)m′(-P-A-P-B)n′(-P-A-S-B)q′(-S-A)r′(-S-B)t′-ORwherein: C is a hydrophobic molecule; A, for each occurrence,independently is a 2′-methoxy-ribonucleotide; B, for each occurrence,independently is a 2′-fluoro-ribonucleotide; L is a linker comprisingone or more moiety selected from the group consisting of: 0-4 repeatunits of ethyleneglycol, a phosphodiester, and a phosphorothioate; S isa phosphorothioate linker; P is a phosphodiester linker; R is selectedfrom hydrogen and a capping group (e.g., an acyl such as acetyl); m′ is0 or 1; n′ is 4, 5 or 6; q′ is 0 or 1; r′ is 0 or 1; and t′ is 0 or 1.

1) Modifications to Enhance Target Discrimination

In certain embodiments, the RNA silencing agents of the invention may besubstituted with a destabilizing nucleotide to enhance single nucleotidetarget discrimination (see U.S. application Ser. No. 11/698,689, filedJan. 25, 2007 and U.S. Provisional Application No. 60/762,225 filed Jan.25, 2006, both of which are incorporated herein by reference). Such amodification may be sufficient to abolish the specificity of the RNAsilencing agent for a non-target mRNA (e.g. wild-type mRNA), withoutappreciably affecting the specificity of the RNA silencing agent for atarget mRNA (e.g. gain-of-function mutant mRNA).

In preferred embodiments, the RNA silencing agents of the invention aremodified by the introduction of at least one universal nucleotide in theantisense strand thereof. Universal nucleotides comprise base portionsthat are capable of base pairing indiscriminately with any of the fourconventional nucleotide bases (e.g. A, G, C, U). A universal nucleotideis preferred because it has relatively minor effect on the stability ofthe RNA duplex or the duplex formed by the guide strand of the RNAsilencing agent and the target mRNA. Exemplary universal nucleotideinclude those having an inosine base portion or an inosine analog baseportion selected from the group consisting of deoxyinosine (e.g.2′-deoxyinosine), 7-deaza-2′-deoxyinosine, 2′-aza-2′-deoxyinosine,PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine,2′-O-methoxyethyl-inosine, and 2′-OMe-inosine. In particularly preferredembodiments, the universal nucleotide is an inosine residue or anaturally occurring analog thereof.

In certain embodiments, the RNA silencing agents of the invention aremodified by the introduction of at least one destabilizing nucleotidewithin 5 nucleotides from a specificity-determining nucleotide (i.e.,the nucleotide which recognizes the disease-related polymorphism). Forexample, the destabilizing nucleotide may be introduced at a positionthat is within 5, 4, 3, 2, or 1 nucleotide(s) from aspecificity-determining nucleotide. In exemplary embodiments, thedestabilizing nucleotide is introduced at a position which is 3nucleotides from the specificity-determining nucleotide (i.e., such thatthere are 2 stabilizing nucleotides between the destabilizing nucleotideand the specificity-determining nucleotide). In RNA silencing agentshaving two strands or strand portions (e.g. siRNAs and shRNAs), thedestabilizing nucleotide may be introduced in the strand or strandportion that does not contain the specificity-determining nucleotide. Inpreferred embodiments, the destabilizing nucleotide is introduced in thesame strand or strand portion that contains the specificity-determiningnucleotide.

2) Modifications to Enhance Efficacy and Specificity

In certain embodiments, the RNA silencing agents of the invention may bealtered to facilitate enhanced efficacy and specificity in mediatingRNAi according to asymmetry design rules (see U.S. Pat. Nos. 8,309,704,7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such alterationsfacilitate entry of the antisense strand of the siRNA (e.g., a siRNAdesigned using the methods of the invention or an siRNA produced from ashRNA) into RISC in favor of the sense strand, such that the antisensestrand preferentially guides cleavage or translational repression of atarget mRNA, and thus increasing or improving the efficiency of targetcleavage and silencing. Preferably the asymmetry of an RNA silencingagent is enhanced by lessening the base pair strength between theantisense strand 5′ end (AS 5′) and the sense strand 3′ end (S 3′) ofthe RNA silencing agent relative to the bond strength or base pairstrength between the antisense strand 3′ end (AS 3′) and the sensestrand 5′ end (S ′5) of said RNA silencing agent.

In one embodiment, the asymmetry of an RNA silencing agent of theinvention may be enhanced such that there are fewer G:C base pairsbetween the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion than between the 3′ end of the first orantisense strand and the 5′ end of the sense strand portion. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one mismatched base pair betweenthe 5′ end of the first or antisense strand and the 3′ end of the sensestrand portion. Preferably, the mismatched base pair is selected fromthe group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one wobble base pair, e.g., G:U,between the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion. In another embodiment, the asymmetry of an RNAsilencing agent of the invention may be enhanced such that there is atleast one base pair comprising a rare nucleotide, e.g., inosine (I).Preferably, the base pair is selected from the group consisting of anI:A, I:U and I:C. In yet another embodiment, the asymmetry of an RNAsilencing agent of the invention may be enhanced such that there is atleast one base pair comprising a modified nucleotide. In preferredembodiments, the modified nucleotide is selected from the groupconsisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

3) RNA Silencing Agents with Enhanced Stability

The RNA silencing agents of the present invention can be modified toimprove stability in serum or in growth medium for cell cultures. Inorder to enhance the stability, the 3′-residues may be stabilizedagainst degradation, e.g., they may be selected such that they consistof purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect the efficiency of RNA interference.

In a preferred aspect, the invention features RNA silencing agents thatinclude first and second strands wherein the second strand and/or firststrand is modified by the substitution of internal nucleotides withmodified nucleotides, such that in vivo stability is enhanced ascompared to a corresponding unmodified RNA silencing agent. As definedherein, an “internal” nucleotide is one occurring at any position otherthan the 5′ end or 3′ end of nucleic acid molecule, polynucleotide oroligonucleotide. An internal nucleotide can be within a single-strandedmolecule or within a strand of a duplex or double-stranded molecule. Inone embodiment, the sense strand and/or antisense strand is modified bythe substitution of at least one internal nucleotide. In anotherembodiment, the sense strand and/or antisense strand is modified by thesubstitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. Inanother embodiment, the sense strand and/or antisense strand is modifiedby the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of theinternal nucleotides. In yet another embodiment, the sense strand and/orantisense strand is modified by the substitution of all of the internalnucleotides.

In a preferred embodiment of the present invention, the RNA silencingagents may contain at least one modified nucleotide analogue. The one ormore nucleotide analogues may be located at positions where thetarget-specific silencing activity, e.g., the RNAi mediating activity ortranslational repression activity is not substantially effected, e.g.,in a region at the 5′-end and/or the 3′-end of the siRNA molecule.Particularly, the ends may be stabilized by incorporating modifiednucleotide analogues.

Exemplary nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In exemplary backbone-modified ribonucleotides, the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In exemplary sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

In particular embodiments, the modifications are 2′-fluoro, 2′-aminoand/or 2′-thio modifications. Particularly preferred modificationsinclude 2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine,2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine,2′-amino-adenosine, 2′-amino-guanosine, 2,6-diaminopurine,4-thio-uridine, and/or 5-amino-allyl-uridine. In a particularembodiment, the 2′-fluoro ribonucleotides are every uridine andcytidine. Additional exemplary modifications include 5-bromo-uridine,5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine,2′-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and5-fluoro-uridine. 2′-deoxy-nucleotides and 2′-Ome nucleotides can alsobe used within modified RNA-silencing agents moities of the instantinvention. Additional modified residues include, deoxy-abasic, inosine,N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purineribonucleoside and ribavirin. In a particularly preferred embodiment,the 2′ moiety is a methyl group such that the linking moiety is a2′-O-methyl oligonucleotide.

In an exemplary embodiment, the RNA silencing agent of the inventioncomprises Locked Nucleic Acids (LNAs). LNAs comprise sugar-modifiednucleotides that resist nuclease activities (are highly stable) andpossess single nucleotide discrimination for mRNA (Elmen et al., NucleicAcids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). Thesemolecules have 2′-O,4′-C-ethylene-bridged nucleic acids, with possiblemodifications such as 2′-deoxy-2″-fluorouridine. Moreover, LNAs increasethe specificity of oligonucleotides by constraining the sugar moietyinto the 3′-endo conformation, thereby pre-organizing the nucleotide forbase pairing and increasing the melting temperature of theoligonucleotide by as much as 10° C. per base.

In another exemplary embodiment, the RNA silencing agent of theinvention comprises Peptide Nucleic Acids (PNAs). PNAs comprise modifiednucleotides in which the sugar-phosphate portion of the nucleotide isreplaced with a neutral 2-amino ethylglycine moiety capable of forming apolyamide backbone which is highly resistant to nuclease digestion andimparts improved binding specificity to the molecule (Nielsen, et al.,Science, (2001), 254: 1497-1500).

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

In other embodiments, cross-linking can be employed to alter thepharmacokinetics of the RNA silencing agent, for example, to increasehalf-life in the body. Thus, the invention includes RNA silencing agentshaving two complementary strands of nucleic acid, wherein the twostrands are crosslinked. The invention also includes RNA silencingagents which are conjugated or unconjugated (e.g., at its 3′ terminus)to another moiety (e.g. a non-nucleic acid moiety such as a peptide), anorganic compound (e.g., a dye), or the like). Modifying siRNAderivatives in this way may improve cellular uptake or enhance cellulartargeting activities of the resulting siRNA derivative as compared tothe corresponding siRNA, are useful for tracing the siRNA derivative inthe cell, or improve the stability of the siRNA derivative compared tothe corresponding siRNA.

Other exemplary modifications include: (a) 2′ modification, e.g.,provision of a 2′ OMe moiety on a U in a sense or antisense strand, butespecially on a sense strand, and/or a 2′ F moiety on a U in a sense orantisense strand, but especially on a sense strand, and/or a 2′ OMemoiety in a 3′ overhang, e.g., at the 3′ terminus (3′ terminus means atthe 3′ atom of the molecule or at the most 3′ moiety, e.g., the most 3′P or 2′ position, as indicated by the context) and/or a 2′ F moiety; (b)modification of the backbone, e.g., with the replacement of an 0 with anS, in the phosphate backbone, e.g., the provision of a phosphorothioatemodification, on the U or the A or both, especially on an antisensestrand; e.g., with the replacement of a P with an S; (c) replacement ofthe U with a C5 amino linker; (d) replacement of an A with a G (sequencechanges are preferred to be located on the sense strand and not theantisense strand); and (d) modification at the 2′, 6′, 7′, or 8′position. Exemplary embodiments are those in which one or more of thesemodifications are present on the sense but not the antisense strand, orembodiments where the antisense strand has fewer of such modifications.Yet other exemplary modifications include the use of a methylated P in a3′ overhang, e.g., at the 3′ terminus; combination of a 2′ modification,e.g., provision of a 2′ O Me moiety and modification of the backbone,e.g., with the replacement of a P with an S, e.g., the provision of aphosphorothioate modification, or the use of a methylated P, in a 3′overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl;modification with an abasic pyrrolidone in a 3′ overhang, e.g., at the3′ terminus; modification with naproxen, ibuprofen, or other moietieswhich inhibit degradation at the 3′ terminus.

4) Modifications to Enhance Cellular Uptake

In other embodiments, RNA silencing agents may be modified with chemicalmoieties, for example, to enhance cellular uptake by target cells (e.g.,neuronal cells). Thus, the invention includes RNA silencing agents whichare conjugated or unconjugated (e.g., at its 3′ terminus) to anothermoiety (e.g. a non-nucleic acid moiety such as a peptide), an organiccompound (e.g., a dye), or the like. The conjugation can be accomplishedby methods known in the art, e.g., using the methods of Lambert et al.,Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loadedto polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J.Control Release 53(1-3):137-43 (1998) (describes nucleic acids bound tonanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994)(describes nucleic acids linked to intercalating agents, hydrophobicgroups, polycations or PACA nanoparticles); and Godard et al., Eur. J.Biochem. 232(2):404-10 (1995) (describes nucleic acids linked tonanoparticles).

In a particular embodiment, an RNA silencing agent of invention isconjugated to a lipophilic moiety. In one embodiment, the lipophilicmoiety is a ligand that includes a cationic group. In anotherembodiment, the lipophilic moiety is attached to one or both strands ofan siRNA. In an exemplary embodiment, the lipophilic moiety is attachedto one end of the sense strand of the siRNA. In another exemplaryembodiment, the lipophilic moiety is attached to the 3′ end of the sensestrand. In certain embodiments, the lipophilic moiety is selected fromthe group consisting of cholesterol, vitamin E, vitamin K, vitamin A,folic acid, or a cationic dye (e.g., Cy3). In an exemplary embodiment,the lipophilic moiety is a cholesterol. Other lipophilic moietiesinclude cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

5) Tethered Ligands

Other entities can be tethered to an RNA silencing agent of theinvention. For example, a ligand tethered to an RNA silencing agent toimprove stability, hybridization thermodynamics with a target nucleicacid, targeting to a particular tissue or cell-type, or cellpermeability, e.g., by an endocytosis-dependent or -independentmechanism. Ligands and associated modifications can also increasesequence specificity and consequently decrease off-site targeting. Atethered ligand can include one or more modified bases or sugars thatcan function as intercalators. These are preferably located in aninternal region, such as in a bulge of RNA silencing agent/targetduplex. The intercalator can be an aromatic, e.g., a polycyclic aromaticor heterocyclic aromatic compound. A polycyclic intercalator can havestacking capabilities, and can include systems with 2, 3, or 4 fusedrings. The universal bases described herein can be included on a ligand.In one embodiment, the ligand can include a cleaving group thatcontributes to target gene inhibition by cleavage of the target nucleicacid. The cleaving group can be, for example, a bleomycin (e.g.,bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline(e.g., O-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr-lystripeptide), or metal ion chelating group. The metal ion chelating groupcan include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II)2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, oracridine, which can promote the selective cleavage of target RNA at thesite of the bulge by free metal ions, such as Lu(III). In someembodiments, a peptide ligand can be tethered to a RNA silencing agentto promote cleavage of the target RNA, e.g., at the bulge region. Forexample, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) canbe conjugated to a peptide (e.g., by an amino acid derivative) topromote target RNA cleavage. A tethered ligand can be an aminoglycosideligand, which can cause an RNA silencing agent to have improvedhybridization properties or improved sequence specificity. Exemplaryaminoglycosides include glycosylated polylysine, galactosylatedpolylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugatesof aminoglycosides, such as Neo-N-acridine, Neo-S-acridine,Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of anacridine analog can increase sequence specificity. For example, neomycinB has a high affinity for RNA as compared to DNA, but lowsequence-specificity. An acridine analog, neo-5-acridine has anincreased affinity for the HIV Rev-response element (RRE). In someembodiments the guanidine analog (the guanidinoglycoside) of anaminoglycoside ligand is tethered to an RNA silencing agent. In aguanidinoglycoside, the amine group on the amino acid is exchanged for aguanidine group. Attachment of a guanidine analog can enhance cellpermeability of an RNA silencing agent. A tethered ligand can be apoly-arginine peptide, peptoid or peptidomimetic, which can enhance thecellular uptake of an oligonucleotide agent.

Exemplary ligands are coupled, preferably covalently, either directly orindirectly via an intervening tether, to a ligand-conjugated carrier. Inexemplary embodiments, the ligand is attached to the carrier via anintervening tether. In exemplary embodiments, a ligand alters thedistribution, targeting or lifetime of an RNA silencing agent into whichit is incorporated. In exemplary embodiments, a ligand provides anenhanced affinity for a selected target, e.g., molecule, cell or celltype, compartment, e.g., a cellular or organ compartment, tissue, organor region of the body, as, e.g., compared to a species absent such aligand.

Exemplary ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified RNA silencing agent, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides. Ligands in general can include therapeuticmodifiers, e.g., for enhancing uptake; diagnostic compounds or reportergroups e.g., for monitoring distribution; cross-linking agents;nuclease-resistance conferring moieties; and natural or unusualnucleobases. General examples include lipophiles, lipids, steroids(e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal),carbohydrates, proteins, protein binding agents, integrin targetingmolecules, polycationics, peptides, polyamines, and peptide mimics.Ligands can include a naturally occurring substance, (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid); amino acid, or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polyaminoacid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quatemary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a placentalcell, a kidney cell and/or a liver cell. A targeting group can be athyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A,mucin carbohydrate, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose,multivalent fucose, glycosylated polyaminoacids, multivalent galactose,transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid,cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or anRGD peptide or RGD peptide mimetic. Other examples of ligands includedyes, intercalating agents (e.g. acridines and substituted acridines),cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lystripeptide, aminoglycosides, guanidium aminoglycodies, artificialendonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol (andthio analogs thereof), cholic acid, cholanic acid, lithocholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters,e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀ fattyacids) and ethers thereof, e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇,C₁₈, C₁₉, or C₂₀ alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol,1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group,hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group,palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, naproxen, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the RNA silencing agent into the cell, for example, bydisrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin. The ligand can increase the uptake of the RNAsilencing agent into the cell by activating an inflammatory response,for example. Exemplary ligands that would have such an effect includetumor necrosis factor alpha (TNFα), interleukin-1 beta, or gammainterferon. In one aspect, the ligand is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue. For example, thetarget tissue can be the placenta, the kidneys or the liver. Othermolecules that can bind HSA can also be used as ligands. For example,neproxin or aspirin can be used. A lipid or lipid-based ligand can (a)increase resistance to degradation of the conjugate, (b) increasetargeting or transport into a target cell or cell membrane, and/or (c)can be used to adjust binding to a serum protein, e.g., HSA. A lipidbased ligand can be used to modulate, e.g., control the binding of theconjugate to a target tissue. For example, a lipid or lipid-based ligandthat binds to HSA more strongly will be less likely to be targeted tothe placenta, liver and/or kidney and therefore less likely to becleared from the body. A lipid or lipid-based ligand that binds to HSAless strongly can be used to target the conjugate to the placenta, liverand/or kidney. Other moieties that target to placental, liver and/orkidney cells can also be used in place of or in addition to the lipidbased ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics tooligonucleotide agents can affect pharmacokinetic distribution of theRNA silencing agent, such as by enhancing cellular recognition andabsorption. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. The peptide moiety can be an L-peptide orD-peptide. In another alternative, the peptide moiety can include ahydrophobic membrane translocation sequence (MTS). A peptide orpeptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature354:82-84, 1991). In exemplary embodiments, the peptide orpeptidomimetic tethered to an RNA silencing agent via an incorporatedmonomer unit is a cell targeting peptide such as anarginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptidemoiety can range in length from about 5 amino acids to about 40 aminoacids. The peptide moieties can have a structural modification, such asto increase stability or direct conformational properties. Any of thestructural modifications described below can be utilized.

6) Combinations

In one aspect, provided herein is a combination comprising:

an oligonucleotide of Formula (I):

(I) (5′-3′) R¹R³X¹[(L²)(X¹)]₁₇R³X¹;an oligonucleotide of Formula (II):

(II) (3′-5′) R²LX²R³X²[(L²)(X²)]₁₂R³X²;an oligonucleotide of Formula (III):

(III) (5′-3′) R¹R³X³[(L²)(X³)]₁₇R³X³;andan oligonucleotide of Formula (IV):

(IV) (5′-3′) R²LX⁴R³X⁴[(L²)(X⁴)]₁₂R³X⁴;or a pharmaceutically acceptable salt thereof,whereinthe oligonucleotide sequence of Formula (I) is different than theoligonucleotide sequence of Formula (III);the oligonucleotide sequence of Formula (II) is different than theoligonucleotide sequence of Formula (IV);R¹ is independently selected at each occurrence from the groupconsisting of

R² is selected from the group consisting of an alkyl chain (e.g., C₁₋₆,C₁₋₁₀, C₁₋₂₀, C₁₋₃₀, or C₁₋₄₀), a vitamin, a ligand, a peptide, abioactive conjugate (including, but not limited to glycosphingolipids,polyunsaturated fatty acids, secosteroids, steroid hormones, or sterollipids),

R³ is independently selected at each occurrence from the groupconsisting of an internucleotide linker as shown in FIG. 41;L is a linker connecting two moieties, wherein the linker is selectedfrom the group consisting of an ethylene glycol chain, an alkyl chain, apeptide, an RNA, a DNA,

and combinations thereof;L² is independently selected at each occurrence from the groupconsisting of internucleotide linkages as shown in FIG. 42; andX¹, X², X³, and X⁴ are each independently selected at each occurrencefrom the group consisting of nucleosides as shown in FIG. 43, whereinthe nucleoside base is selected from the group consisting of adenine,guanine, uracil, cytosine, 5-methylcytosine, hypoxanthine, and thymine,wherein the nucleoside base is optionally further modified with one ormore additional hydrophobic moieties selected from naphthyl or isobutyl.

In one embodiment, R¹ is selected from the group consisting of

In another embodiment, R¹ is

In another embodiment, R² is selected from the group consisting of

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, a methylphosphonate, amethylenephosphonate, a phosphotriester, and a boranophosphate.

In another embodiment, R³ is an internucleotide linker independentlyselected at each occurrence from the group consisting of aphosphorothioate, a phosphorodithioate, and a boranophosphate.

In another embodiment, R³ is a phosphorothioate.

In another embodiment, L is selected from the group consisting of anethylene glycol chain, an alkyl chain, and a peptide.

In another embodiment, L is selected from an ethylene glycol chain or apeptide.

In yet another embodiment, L is

In still another embodiment, L is

In another embodiment, L is

In one embodiment, L² is independently selected at each occurrence froma phosphodiester and a phosphorothioate.

In one embodiment, X¹, X², X³, and X⁴ are each independently selected ateach occurrence from the group consisting of nucleosides as shown inFIG. 43, wherein the nucleoside base is selected from the groupconsisting of adenine, guanine, uracil, cytosine, 5-methylcytosine,hypoxanthine, and thymine.

In one embodiment, X¹, X², X³, and X⁴ are each independently selected ateach occurrence from

wherein the nucleoside base is selected from the group consisting ofadenine, guanine, uracil, cytosine, 5-methylcytosine, hypoxanthine, andthymine.

In one embodiment, the combination is a combination shown in FIG. 39, ora pharmaceutically acceptable salt thereof.

In one embodiment, the combination is a combination shown in FIG. 39, ora pharmaceutically acceptable salt thereof, wherein

R¹ is

R² is

In another embodiment, the combination is a combination shown in FIG.39, or a pharmaceutically acceptable salt thereof, wherein

R¹ is

R² is

In one embodiment, the combination is a combination shown in FIG. 40, ora pharmaceutically acceptable salt thereof.

In one embodiment, the combination is a combination shown in FIG. 40, ora pharmaceutically acceptable salt thereof, wherein

R¹ is

R² is

In another embodiment, the combination is a combination shown in FIG.40, or a pharmaceutically acceptable salt thereof, wherein

R¹ is

R² is

V. Methods of Introducing Nucleic Acids, Vectors and Host Cells

RNA silencing agents of the invention may be directly introduced intothe cell (e.g., a neural cell) (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, or may be introduced by bathing acell or organism in a solution containing the nucleic acid. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the nucleic acid may be introduced.

The RNA silencing agents of the invention can be introduced usingnucleic acid delivery methods known in art including injection of asolution containing the nucleic acid, bombardment by particles coveredby the nucleic acid, soaking the cell or organism in a solution of thenucleic acid, or electroporation of cell membranes in the presence ofthe nucleic acid. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and cationic liposome transfection such ascalcium phosphate, and the like. The nucleic acid may be introducedalong with other components that perform one or more of the followingactivities: enhance nucleic acid uptake by the cell or other-wiseincrease inhibition of the target gene.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or other-wise increase inhibition of thetarget gene.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing a cell or organism in a solution containing the RNA. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target gene and the dose of double strandedRNA material delivered, this process may provide partial or completeloss of function for the target gene. A reduction or loss of geneexpression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more oftargeted cells is exemplary. Inhibition of gene expression refers to theabsence (or observable decrease) in the level of protein and/or mRNAproduct from a target gene. Specificity refers to the ability to inhibitthe target gene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, Enzyme Linked ImmunoSorbent Assay (ELISA), Western blotting,RadiolmmunoAssay (RIA), other immunoassays, and Fluorescence ActivatedCell Sorting (FACS).

For RNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of RNAi agent may result in inhibitionin a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%,or 95% of targeted cells). Quantization of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell; mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

The RNA may be introduced in an amount which allows delivery of at leastone copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of material may yield more effective inhibition; lowerdoses may also be useful for specific applications.

In an exemplary aspect, the efficacy of an RNAi agent of the invention(e.g., an siRNA targeting an flt1 intronic target sequence) is testedfor its ability to specifically degrade mutant mRNA (e.g., sflt1 mRNAand/or the production of sFlt1 protein) in cells, in particular, inplacental cells (e.g., labyrinth cells, trophoblasts (e.g.,syncytiotrophoblasts and/or cytotrophoblasts), mesenchymal cells,mesenchymal-derived macrophages (Hofbauer cells), fibroblasts, fetalvascular cells (e.g., smooth muscle cells, perivascular cells(pericytes), and endothelial cells)), liver cells and/or kidney cells.Also suitable for cell-based validation assays are other readilytransfectable cells, for example, trophoblast cells, HeLa cells or COScells. Cells are transfected with human wild type or mutant cDNAs (e.g.,human wild-type or secreted flt1 cDNA). Standard siRNA, modified siRNAor vectors able to produce siRNA from U-looped mRNA are co-transfected.Selective reduction in target mRNA (e.g., sflt1 mRNA) and/or targetprotein (e.g., sFlt1 protein) is measured. Reduction of target mRNA orprotein can be compared to levels of target mRNA or protein in theabsence of an RNAi agent or in the presence of an RNAi agent that doesnot target sFlt1 mRNA. Exogenously-introduced mRNA or protein (orendogenous mRNA or protein) can be assayed for comparison purposes. Whenutilizing neuronal cells, which are known to be somewhat resistant tostandard transfection techniques, it may be desirable to introduce RNAiagents (e.g., siRNAs) by passive uptake.

6) Recombinant Adeno-Associated Viruses and Vectors

In certain exemplary embodiments, recombinant adeno-associated viruses(rAAVs) and their associated vectors can be used to deliver one or moresiRNAs into cells, e.g., placental cells, kidney cells and/or livercells. AAV is able to infect many different cell types, although theinfection efficiency varies based upon serotype, which is determined bythe sequence of the capsid protein. Several native AAV serotypes havebeen identified, with serotypes 1-9 being the most commonly used forrecombinant AAV. AAV-2 is the most well-studied and published serotype.The AAV-DJ system includes serotypes AAV-DJ and AAV-DJ/8. Theseserotypes were created through DNA shuffling of multiple AAV serotypesto produce AAV with hybrid capsids that have improved transductionefficiencies in vitro (AAV-DJ) and in vivo (AAV-DJ/8) in a variety ofcells and tissues.

In particular embodiments, widespread central nervous system (CNS)delivery can be achieved by intravascular delivery of recombinantadeno-associated virus 7 (rAAV7), RAAV9 and rAAV10, or other suitablerAAVs (Zhang et al. (2011) Mol. Ther. 19(8):1440-8. doi:10.1038/mt.2011.98. Epub 2011 May 24). rAAVs and their associatedvectors are well-known in the art and are described in US PatentApplications 2014/0296486, 2010/0186103, 2008/0269149, 2006/0078542 and2005/0220766, each of which is incorporated herein by reference in itsentirety for all purposes.

rAAVs may be delivered to a subject in compositions according to anyappropriate methods known in the art. An rAAV can be suspended in aphysiologically compatible carrier (i.e., in a composition), and may beadministered to a subject, i.e., a host animal, such as a human, mouse,rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig,hamster, chicken, turkey, a non-human primate (e.g., baboon) or thelike. In certain embodiments, a host animal is a non-human host animal.

Delivery of one or more rAAVs to a mammalian subject may be performed,for example, by intramuscular injection or by administration into thebloodstream of the mammalian subject. Administration into thebloodstream may be by injection into a vein, an artery, or any othervascular conduit. In certain embodiments, one or more rAAVs areadministered into the bloodstream by way of isolated limb perfusion, atechnique well known in the surgical arts, the method essentiallyenabling the artisan to isolate a limb from the systemic circulationprior to administration of the rAAV virions. A variant of the isolatedlimb perfusion technique, described in U.S. Pat. No. 6,177,403, can alsobe employed by the skilled artisan to administer virions into thevasculature of an isolated limb to potentially enhance transduction intomuscle cells or tissue. Moreover, in certain instances, it may bedesirable to deliver virions to the placenta, liver and/or kidneys of asubject. Recombinant AAVs may be delivered directly to the placenta,liver and/or kidney with a needle, catheter or related device, usingneurosurgical techniques known in the art, such as by stereotacticinjection (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidsonet al., PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223,1993; and Alisky and Davidson, Hum. Gene Ther. 11:2315-2329, 2000).

The compositions of the invention may comprise an rAAV alone, or incombination with one or more other viruses (e.g., a second rAAV encodinghaving one or more different transgenes). In certain embodiments, acomposition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentrAAVs each having one or more different transgenes.

An effective amount of an rAAV is an amount sufficient to target infectan animal, target a desired tissue. In some embodiments, an effectiveamount of an rAAV is an amount sufficient to produce a stable somatictransgenic animal model. The effective amount will depend primarily onfactors such as the species, age, weight, health of the subject, and thetissue to be targeted, and may thus vary among animal and tissue. Forexample, an effective amount of one or more rAAVs is generally in therange of from about 1 ml to about 100 ml of solution containing fromabout 10⁹ to 10¹⁶ genome copies. In some cases, a dosage between about10¹¹ to 10¹² rAAV genome copies is appropriate. In certain embodiments,10¹² rAAV genome copies is effective to target heart, liver, andpancreas tissues. In some cases, stable transgenic animals are producedby multiple doses of an rAAV.

In some embodiments, rAAV compositions are formulated to reduceaggregation of AAV particles in the composition, particularly where highrAAV concentrations are present (e.g., about 10¹³ genome copies/mL ormore). Methods for reducing aggregation of rAAVs are well known in theart and, include, for example, addition of surfactants, pH adjustment,salt concentration adjustment, etc. (See, e.g., Wright et al. (2005)Molecular Therapy 12:171-178, the contents of which are incorporatedherein by reference.)

“Recombinant AAV (rAAV) vectors” comprise, at a minimum, a transgene andits regulatory sequences, and 5′ and 3′ AAV inverted terminal repeats(ITRs). It is this recombinant AAV vector which is packaged into acapsid protein and delivered to a selected target cell. In someembodiments, the transgene is a nucleic acid sequence, heterologous tothe vector sequences, which encodes a polypeptide, protein, functionalRNA molecule (e.g., siRNA) or other gene product, of interest. Thenucleic acid coding sequence is operatively linked to regulatorycomponents in a manner which permits transgene transcription,translation, and/or expression in a cell of a target tissue.

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are usually about 145 basepairs in length. Incertain embodiments, substantially the entire sequences encoding theITRs are used in the molecule, although some degree of minormodification of these sequences is permissible. The ability to modifythese ITR sequences is within the skill of the art. (See, e.g., textssuch as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2ded., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher etal., J Virol., 70:520 532 (1996)). An example of such a moleculeemployed in the present invention is a “cis-acting” plasmid containingthe transgene, in which the selected transgene sequence and associatedregulatory elements are flanked by the 5′ and 3′ AAV ITR sequences. TheAAV ITR sequences may be obtained from any known AAV, includingmammalian AAV types described further herein.

VI. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a diseaseor disorder caused, in whole or in part, by secreted Flt1 protein. Inone embodiment, the disease or disorder is a liver disease or disorder.In another embodiment, the disease or disorder is a kidney disease ordisorder. In one embodiment, the disease or disorder is a placentaldisease or disorder. In one embodiment, the disease or disorder is apregnancy-related disease or disorder. In a preferred embodiment, thedisease or disorder is a disorder associated with the expression ofsoluble Flt1 protein and in which amplified expression of the solubleFlt1 protein leads to clinical manifestations of PE, postpartum PE,eclampsia and/or HELLP.

“Treatment,” or “treating,” as used herein, is defined as theapplication or administration of a therapeutic agent (e.g., a RNA agentor vector or transgene encoding same) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has the disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder as described above, by administering tothe subject a therapeutic agent (e.g., an RNAi agent or vector ortransgene encoding same). Subjects at risk for the disease can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe disease or disorder, such that the disease or disorder is preventedor, alternatively, delayed in its progression.

Another aspect of the invention pertains to methods treating subjectstherapeutically, i.e., alter onset of symptoms of the disease ordisorder. In an exemplary embodiment, the modulatory method of theinvention involves contacting a cell expressing a gain-of-functionmutant with a therapeutic agent (e.g., a RNAi agent or vector ortransgene encoding same) that is specific for one or more targetsequences within the gene (e.g., SEQ ID NOs:1, 2, 3 or 4 or anycombinations thereof), such that sequence specific interference with thegene is achieved. These methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject).

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics,” as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype,” or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

Therapeutic agents can be tested in an appropriate animal model. Forexample, an RNAi agent (or expression vector or transgene encoding same)as described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with said agent.Alternatively, a therapeutic agent can be used in an animal model todetermine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

A pharmaceutical composition containing an RNA silencing agent of theinvention can be administered to any patient diagnosed as having or atrisk for developing a pregnancy-, liver- and/or kidney-related disorder,such as PE and/or eclampsia. In one embodiment, the patient is diagnosedas having a PE and/or eclampsia, and the patient is otherwise in generalgood health. For example, the patient is not terminally ill, and thepatient is likely to live at least 2, 3, 5 or more years followingdiagnosis. The patient can be treated immediately following diagnosis,or treatment can be delayed until the patient is experiencing moredebilitating symptoms, such as two or more symptoms of PE or one or moresymptoms of eclampsia. In another embodiment, the patient has notreached an advanced stage of the disease.

An RNA silencing agent modified for enhance uptake into neural cells canbe administered at a unit dose less than about 1.4 mg per kg ofbodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, andless than 200 nmole of RNA agent (e.g., about 4.4×10¹⁶ copies) per kg ofbodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75,0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNAsilencing agent per kg of bodyweight. The unit dose, for example, can beadministered by injection (e.g., intravenous or intramuscular,intrathecally, or directly into the brain), an inhaled dose, or atopical application. Particularly preferred dosages are less than 2, 1,or 0.1 mg/kg of body weight.

Delivery of an RNA silencing agent directly to an organ (e.g., directlyto the placenta, liver and/or kidneys) can be at a dosage on the orderof about 0.00001 mg to about 3 mg per organ, or preferably about0.0001-0.001 mg per organ, about 0.03-3.0 mg per organ, about 0.1-3.0 mgper eye or about 0.3-3.0 mg per organ. The dosage can be an amounteffective to treat or prevent a liver-, kidney- or pregnancy-relateddisease or disorder, e.g., PE, postpartum PE, eclampsia and/or HELLP. Inone embodiment, the unit dose is administered less frequently than oncea day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment,the unit dose is not administered with a frequency (e.g., not a regularfrequency). For example, the unit dose may be administered a singletime. In one embodiment, the effective dose is administered with othertraditional therapeutic modalities.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an RNA silencing agent. The maintenance doseor doses are generally lower than the initial dose, e.g., one-half lessof the initial dose. A maintenance regimen can include treating thesubject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of bodyweight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg ofbodyweight per day. The maintenance doses are preferably administered nomore than once every 5, 10, or 30 days. Further, the treatment regimenmay last for a period of time which will vary depending upon the natureof the particular disease, its severity and the overall condition of thepatient. In preferred embodiments the dosage may be delivered no morethan once per day, e.g., no more than once per 24, 36, 48, or morehours, e.g., no more than once every 5 or 8 days. Following treatment,the patient can be monitored for changes in his condition and foralleviation of the symptoms of the disease state. The dosage of thecompound may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disease state isobserved, if the disease state has been ablated, or if undesiredside-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracistemal or intracapsular), orreservoir may be advisable. In one embodiment, a pharmaceuticalcomposition includes a plurality of RNA silencing agent species. Inanother embodiment, the RNA silencing agent species has sequences thatare non-overlapping and non-adjacent to another species with respect toa naturally occurring target sequence. In another embodiment, theplurality of RNA silencing agent species is specific for differentnaturally occurring target genes. In another embodiment, the RNAsilencing agent is allele specific. In another embodiment, the pluralityof RNA silencing agent species target two or more target sequences(e.g., two, three, four, five, six, or more target sequences).

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the RNA silencing agent composition is an amountsufficient to be effective in treating or preventing a disorder or toregulate a physiological condition in humans. The concentration oramount of RNA silencing agent administered will depend on the parametersdetermined for the agent and the method of administration, e.g. nasal,buccal, or pulmonary. For example, nasal formulations tend to requiremuch lower concentrations of some ingredients in order to avoidirritation or burning of the nasal passages. It is sometimes desirableto dilute an oral formulation up to 10-100 times in order to provide asuitable nasal formulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an RNA silencing agent caninclude a single treatment or, preferably, can include a series oftreatments. It will also be appreciated that the effective dosage of anRNA silencing agent for treatment may increase or decrease over thecourse of a particular treatment. Changes in dosage may result andbecome apparent from the results of diagnostic assays as describedherein. For example, the subject can be monitored after administering anRNA silencing agent composition. Based on information from themonitoring, an additional amount of the RNA silencing agent compositioncan be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g., a gene thatproduces a target RNA, e.g., an RNA expressed in a liver, kidney and/orplacental cell. The transgenic animal can be deficient for thecorresponding endogenous RNA. In another embodiment, the composition fortesting includes an RNA silencing agent that is complementary, at leastin an internal region, to a sequence that is conserved between thetarget RNA in the animal model and the target RNA in a human.

VII. Pharmaceutical Compositions and Methods of Administration

The invention pertains to uses of the above-described agents forprophylactic and/or therapeutic treatments as described Infra.Accordingly, the modulators (e.g., RNAi agents) of the present inventioncan be incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, antibody, or modulatory compound and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous (IV),intradermal, subcutaneous (SC or SQ), intraperitoneal, intramuscular,oral (e.g., inhalation), transdermal (topical), and transmucosaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The RNA silencing agents can also be administered by transfection orinfection using methods known in the art, including but not limited tothe methods described in McCaffrey et al. (2002), Nature, 418(6893),38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol.,20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J.Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst.Pharm. 53(3), 325 (1996).

The RNA silencing agents can also be administered by any method suitablefor administration of nucleic acid agents, such as a DNA vaccine. Thesemethods include gene guns, bio injectors, and skin patches as well asneedle-free methods such as the micro-particle DNA vaccine technologydisclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermalneedle-free vaccination with powder-form vaccine as disclosed in U.S.Pat. No. 6,168,587. Additionally, intranasal delivery is possible, asdescribed in, inter alia, Hamajima et al. (1998), Clin. Immunol.Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat.No. 6,472,375) and microencapsulation can also be used. Biodegradabletargetable microparticle delivery systems can also be used (e.g., asdescribed in U.S. Pat. No. 6,471,996).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Althoughcompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack ordispenser together with optional instructions for administration.

As defined herein, a therapeutically effective amount of a RNA silencingagent (i.e., an effective dosage) depends on the RNA silencing agentselected. For instance, if a plasmid encoding shRNA is selected, singledose amounts in the range of approximately 1 μg to 1000 mg may beadministered; in some embodiments, 10, 30, 100 or 1000 μg may beadministered. In some embodiments, 1-5 g of the compositions can beadministered. The compositions can be administered one from one or moretimes per day to one or more times per week; including once every otherday. The skilled artisan will appreciate that certain factors mayinfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a protein, polypeptide, or antibodycan include a single treatment or, preferably, can include a series oftreatments.

The nucleic acid molecules of the invention can be inserted intoexpression constructs, e.g., viral vectors, retroviral vectors,expression cassettes, or plasmid viral vectors, e.g., using methodsknown in the art, including but not limited to those described in Xia etal., (2002), Supra. Expression constructs can be delivered to a subjectby, for example, inhalation, orally, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91,3054-3057). The pharmaceutical preparation of the delivery vector caninclude the vector in an acceptable diluent, or can comprise a slowrelease matrix in which the delivery vehicle is imbedded. Alternatively,where the complete delivery vector can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can include one or more cells which produce the genedelivery system.

The nucleic acid molecules of the invention can also include smallhairpin RNAs (shRNAs), and expression constructs engineered to expressshRNAs. Transcription of shRNAs is initiated at a polymerase III (polIII) promoter, and is thought to be terminated at position 2 of a4-5-thymine transcription termination site. Upon expression, shRNAs arethought to fold into a stem-loop structure with 3′ UU-overhangs;subsequently, the ends of these shRNAs are processed, converting theshRNAs into siRNA-like molecules of about 21 nucleotides. Brummelkamp etal. (2002), Science, 296, 550-553; Lee et al, (2002). supra; Miyagishiand Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al.(2002), supra; Paul (2002), supra; Sui (2002) supra; Yu et al. (2002),supra.

The expression constructs may be any construct suitable for use in theappropriate expression system and include, but are not limited toretroviral vectors, linear expression cassettes, plasmids and viral orvirally-derived vectors, as known in the art. Such expression constructsmay include one or more inducible promoters, RNA Pol III promotersystems such as U6 snRNA promoters or H1 RNA polymerase III promoters,or other promoters known in the art. The constructs can include one orboth strands of the siRNA. Expression constructs expressing both strandscan also include loop structures linking both strands, or each strandcan be separately transcribed from separate promoters within the sameconstruct. Each strand can also be transcribed from a separateexpression construct, Tuschl (2002), Supra.

The route of delivery can be dependent on the disorder of the patient.In certain exemplary embodiments, a subject diagnosed with PE,postpartum PE, eclampsia and/or HELLP can be administered an anti-sFlt1RNA silencing agent of the invention by IV or SC administration. Inaddition to an RNA silencing agent of the invention, a patient can beadministered a second therapy, e.g., a palliative therapy and/ordisease-specific therapy. The secondary therapy can be, for example,symptomatic (e.g., for alleviating symptoms), protective (e.g., forslowing or halting disease progression), or restorative (e.g., forreversing the disease process). For the treatment of PE, postpartum PE,eclampsia and/or HELLP, for example, symptomatic therapies can furtherinclude the drugs Atenolol, Hydralazine, Labetalol, magnesium sulfate,Methyldopa, Nicardipine, Nifedipine, sodium nitroprusside and the like.

In general, an RNA silencing agent of the invention can be administeredby any suitable method. As used herein, topical delivery can refer tothe direct application of an RNA silencing agent to any surface of thebody, including the eye, a mucous membrane, surfaces of a body cavity,or to any internal surface. Formulations for topical administration mayinclude transdermal patches, ointments, lotions, creams, gels, drops,sprays, and liquids. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Topical administration can also be used as a means toselectively deliver the RNA silencing agent to the epidermis or dermisof a subject, or to specific strata thereof, or to an underlying tissue.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives. Compositions for intrathecal orintraventricular administration preferably do not include a transfectionreagent or an additional lipophilic moiety besides, for example, thelipophilic moiety attached to the RNA silencing agent.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

An RNA silencing agent of the invention can be administered to a subjectby pulmonary delivery. Pulmonary delivery compositions can be deliveredby inhalation of a dispersion so that the composition within thedispersion can reach the lung where it can be readily absorbed throughthe alveolar region directly into blood circulation. Pulmonary deliverycan be effective both for systemic delivery and for localized deliveryto treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self-contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. An RNAsilencing agent composition may be stably stored as lyophilized orspray-dried powders by itself or in combination with suitable powdercarriers. The delivery of a composition for inhalation can be mediatedby a dosing timing element which can include a timer, a dose counter,time measuring device, or a time indicator which when incorporated intothe device enables dose tracking, compliance monitoring, and/or dosetriggering to a patient during administration of the aerosol medicament.

The types of pharmaceutical excipients that are useful as carriersinclude stabilizers such as Human Serum Albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin; and polysaccharides, such as raffinose,maltodextrins, dextrans, and the like; alditols, such as mannitol,xylitol, and the like. A preferred group of carbohydrates includeslactose, trehalose, raffinose maltodextrins, and mannitol. Suitablepolypeptides include aspartame. Amino acids include alanine and glycine,with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

An RNA silencing agent of the invention can be administered by oral andnasal delivery. For example, drugs administered through these membraneshave a rapid onset of action, provide therapeutic plasma levels, avoidfirst pass effect of hepatic metabolism, and avoid exposure of the drugto the hostile gastrointestinal (GI) environment. Additional advantagesinclude easy access to the membrane sites so that the drug can beapplied, localized and removed easily. In one embodiment, an RNAsilencing agent administered by oral or nasal delivery has been modifiedto be capable of traversing the blood-brain barrier.

In one embodiment, unit doses or measured doses of a composition thatinclude RNA silencing agents are dispensed by an implanted device. Thedevice can include a sensor that monitors a parameter within a subject.For example, the device can include a pump, such as an osmotic pump and,optionally, associated electronics.

An RNA silencing agent can be packaged in a viral natural capsid or in achemically or enzymatically produced artificial capsid or structurederived therefrom.

VIII. Kits

In certain other aspects, the invention provides kits that include asuitable container containing a pharmaceutical formulation of an RNAsilencing agent, e.g., a double-stranded RNA silencing agent, or sRNAagent, (e.g., a precursor, e.g., a larger RNA silencing agent which canbe processed into a sRNA agent, or a DNA which encodes an RNA silencingagent, e.g., a double-stranded RNA silencing agent, or sRNA agent, orprecursor thereof). In certain embodiments the individual components ofthe pharmaceutical formulation may be provided in one container.Alternatively, it may be desirable to provide the components of thepharmaceutical formulation separately in two or more containers, e.g.,one container for an RNA silencing agent preparation, and at leastanother for a carrier compound. The kit may be packaged in a number ofdifferent configurations such as one or more containers in a single box.The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods described hereinmay be made using suitable equivalents without departing from the scopeof the embodiments disclosed herein. Having now described certainembodiments in detail, the same will be more clearly understood byreference to the following examples, which are included for purposes ofillustration only and are not intended to be limiting.

EXAMPLES Example 1. Background and Significance of Preeclampsia (PE)

Overwhelming evidence from epidemiological and experimental studies nowindicates that PE is caused by elevated levels of “soluble decoy”proteins (soluble FLT1s (sFLT1s)) from the Flt1 gene (VEGFR1) in themother's blood stream (Young, B. C., Levine, R. J. & Karumanchi, S. A.Pathogenesis of preeclampsia. Annual review of pathology 5, 173-192(2010); Maynard, S. E. et al. Excess placental soluble fms-like tyrosinekinase 1 (sFlt1) may contribute to endothelial dysfunction,hypertension, and proteinuria in preeclampsia. The Journal of clinicalinvestigation 111, 649-658 (2003); Levine, R. J. et al. Circulatingangiogenic factors and the risk of preeclampsia. The New England journalof medicine 350, 672-683 (2004); Heydarian, M. et al. Novel splicevariants of sFlt1 are upregulated in preeclampsia. Placenta 30, 250-255(2009)). FLT1 is a receptor tyrosine kinase (RTK) predominantlyexpressed in the placenta. A general mechanism for RTK modulation isproduction of truncated, secreted forms of the receptor that act asdominant negative regulators of the overall signaling pathway (FIG. 1A).Ligand sequestration by such soluble decoys inhibits intracellularsignaling by the full-length receptor, thereby desensitizing the systemto ligand concentration (Vorlova, S. et al. Induction of antagonisticsoluble decoy receptor tyrosine kinases by intronic polyA activation.Molecular cell 43, 927-939 (2011).). In the case of FLT1, the solubledecoys are expressed from truncated mRNAs generated by polyadenylationwithin two introns (i13 and i15) upstream of the exons encoding thefl-FLT1 transmembrane (TM) and kinase domains (FIG. 1B).

In mammals, FLT1 is predominantly expressed in the placenta, with humanplacental Flt1 mRNA levels being 10-100 times higher than those observedin other adult tissues (Cerdeira, A. S. & Karumanchi, S. A. Angiogenicfactors in preeclampsia and related disorders. Cold Spring Harborperspectives in medicine 2 (2012)). Whereas the full-length isoformpredominates in all tissues in non-pregnant adult humans (Id.),placental expression is dominated by three truncated isoforms, sFlt1-i13short, sFlt1-i13 long and sFlt1-i15a, all of which encode sFLT1 proteins(FIG. 1B). This same pattern of high Flt1 in placenta and low expressionin other non-pregnant adult tissues is observed in rodents. However,because rodents lack the intron 14 polyadenylation site, they onlyexpress a single soluble decoy form: sFlt1-i13. In PE, both full-length(fl-Flt1) and truncated Flt1 mRNAs accumulate to higher levels in theplacenta than in normal pregnancies, with the truncated isoforms beingeven more pronounced (FIG. 1B). These changes at the mRNA level likelyexplain the significant rise in sFLT1 proteins in the maternalbloodstream during PE.

1.1 Applicability of siRNAs for Treatment of PE

siRNA-based therapeutics were designed for the treatment of PE. Bothpreclinical and clinical data support decreasing sFLT1 as a validtherapeutic strategy for prolonging PE pregnancies (Thadhani, R. et al.Pilot study of extracorporeal removal of soluble fms-like tyrosinekinase 1 in preeclampsia. Circulation 124, 940-950 (2011)). Further, theunique region specific to each sFLT1 protein is very small, with only ahandful of unique amino acids being appended to each C-terminus. Thissmall target size hinders development of conventional drugs (e.g., smallmolecules and antibodies) targeting only sFLT1s and not fl-FLT1. On theother hand, the target window at the RNA level is much larger, with thei13 and i15 mRNA isoforms having 435 and 567 unique bases, respectively,neither of which are present in fl-Flt1 mRNA. Because RNAi requires atarget size of only 19-22 nucleotides, this was determined to be morethan sufficient nucleotide space in which to design multipleisoform-selective siRNAs. From a clinical perspective, the possibilitythat a single dose delivered subcutaneously will be sufficient toprevent runaway sFLT1 expression for several weeks could make treatmentsimple and affordable.

Novel chemically-modified oligonucleotides known as self-deliveringhydrophobically modified siRNAs (hsiRNAs) (FIG. 2A) could provide themost significant advantage for a cost effective therapeutic. While theircurrent cost of chemical synthesis ($200 per gram, with approximately$20 per dose at 1 mg/kg dose levels) is relatively high, the price isexpected to decrease dramatically (10-50 fold) with a kg-level scale-up.Further, hsiRNAs can be fully synthesized using solid support chemistryin less than 10 hours. Like other oligonucleotides, dried hsiRNAs arehighly stable, can be stored for extensive time (i.e., years) at ambienttemperature, and can be brought into solution just prior to injection.Further, hsiRNA half-life in vivo is of sufficient duration that asingle intravenous dose is well suited for a two to six week inhibitionof sFLt1 production.

The ONTs that neutralize sFlt1 described herein are the first novelpreeclampsia therapy based on a mechanistic understanding of thedisease, and could be cost-effectively and easily administeredthroughout the world.

1.2 Pilot Product Target Profile for RNAi-Based Treatment of PE.

The table at FIG. 14 summarizes the current view on acceptable and idealtarget product profiles according to preferred embodiments. Specialconsiderations for developing an RNAi-based treatment for PE arediscussed below.

1.3 Multiple sFLT1 mRNA Isoforms

By performing polyadenylation site sequencing (PAS-Seq (Heyer, E. E.,Ozadam, H., Ricci, E. P., Cenik, C. & Moore, M. J. An optimized kit-freemethod for making strand-specific deep sequencing libraries from RNAfragments. Nucleic Acids Res 43, e2 (2015))) on total RNA from multiplenormal and PE placentas, it was determined that PE placentas overexpressi13 and i15 sFLT1 variants with, i15 being responsible for 55% of readsand i13 responsible for approximately 45% of reads (FIG. 1C). Withoutintending to be bound by scientific theory, the intrinsic variability inisoform ratios in different samples indicates that targeting bothisoforms might be the best option to cover the majority of PE patients.Thus, the candidate drug product was defined as an equimolar mixture oftwo hsiRNAs: one targeting both short and long sFLT1-i13 and anothertargeting sFlt1-i15a (FIG. 3). The FDA has already allowed an siRNAmixture to be defined as a single drug entity when the component siRNAsare identically formulated or chemically modified and their PK/PDprofiles are very similar (e.g., multi-siRNA formulations targetingVEGF-A/KSP (Tabernero, J. et al. First-in-humans trial of an RNAinterference therapeutic targeting VEGF and KSP in cancer patients withliver involvement. Cancer discovery 3, 406-417 (2013)); HBV (Wooddell,C. I. et al. Hepatocyte-targeted RNAi Therapeutics for the Treatment ofChronic Hepatitis B Virus Infection. Molecular therapy: the journal ofthe American Society of Gene Therapy 21, 973-985 (2013)), Arrowhead,etc.). Although using a mixture adds complexity to CMC (Chemistry,Manufacturing and Controls), this is outweighed by the advantage thatthe mixture will allow treatment of wider PE populations independent ofisoform variant overexpression ratios. In certain embodiments, a mixtureof two candidates is administrated subcutaneously (SC) in saline as anexcipient.

In certain embodiments, the desired level of sFLT1 silencing is only30-40%, as a higher degree of silencing might be disadvantageous.Preliminary data indicated that a 10-20 mg/kg dose produced >50%silencing in mice, so lesser silencing may simply be achieved with lowerdosing. Because the desired product profile is a one-time injection,however, higher doses might be required to extend effect duration. Thus,in certain embodiments, i13 or i15 may be used alone as a clinicalcandidate.

1.4 Overall Safety and Toxicity Considerations.

ONT-related toxicity can be due to target-specific effects (e.g., toomuch silencing of sFlt1 isoforms), target-independent effects (i.e.,unintentional silencing of non-target mRNAs) or class-relatedchemistry-specific events. The ability to target the i13 and i15variants separately dramatically reduces the chances of any majortarget-related toxicity. Further, the i13 and i15 variants are placenta-and pregnancy-specific, with low or undetectable expression in otheradult tissues. Therefore, clinically limiting toxicity will most likelybe target-independent. These types of effects include siRNAoff-targeting, RNA-based induction of the innate immune response, andgeneral toxicity related to the chosen mode of delivery (e.g.,hydrophobic modifications in combination with phosphorothioates). Themost advanced bioinformatics was employed up-front upfront to optimizeoligonucleotide design to minimize potential off-target events (Uchida,S. et al. An integrated approach for the systematic identification andcharacterization of heart-enriched genes with unknown functions. BMCgenomics 10, 100 (2009)). Further, all riboses in the seed sequence(i.e., nucleotides 2-8 of the guide strand) were 2′-F and 2′-O-methylmodified, which modifications by themselves are well-established tominimize off-target events (Jackson, A. L. et al. Position-specificchemical modification of siRNAs reduces “off-target” transcriptsilencing. Rna 12, 1197-1205 (2006)). While evaluation of off-targetingsignatures could be established in vitro and in mouse samples usingmicroarray profiling (Jackson, A. L. et al. Position-specific chemicalmodification of siRNAs reduces “off-target” transcript silencing. Rna12, 1197-1205 (2006); Anderson, E., Boese, Q., Khvorova, A. & Karpilow,J. Identifying siRNA-induced off-targets by microarray analysis. Methodsin molecular biology 442, 45-63 (2008); Anderson, E. M. et al.Experimental validation of the importance of seed complement frequencyto siRNA specificity. Rna 14, 853-861 (2008); Birmingham, A. et al. 3′UTR seed matches, but not overall identity, are associated with RNAioff-targets. Nat Methods 3, 199-204 (2006); Fedorov, Y. et al.Off-target effects by siRNA can induce toxic phenotype. Rna 12,1188-1196 (2006)), because the overlap between siRNA off-targetingsignatures in tissue culture/animal models and humans is generallyminimal (Burchard, J. et al. MicroRNA-like off-target transcriptregulation by siRNAs is species specific. Rna 15, 308-315 (2009)), thevalue of such studies is questionable. For each sFLT1 isoform, twodifferent sequences were selected for in vivo evaluation (one lead andone back-up) (FIG. 3). If the lead fails due to off-targeting-inducedtoxicity, the second sequence will be used as a backup (Jackson, A. L. &Linsley, P. S. Recognizing and avoiding siRNA off-target effects fortarget identification and therapeutic application. Nature reviews. Drugdiscovery 9, 57-67 (2010)). As there is currently no formal guidancespecific to siRNA therapeutics, the standard recommendation for NCE (NewChemical Entity) development, including demonstrating safety in twoanimal models (Hughes M, I. J., Kurtz A, et al. (ed. C. N. Sittampalam GS, Nelson H, et al., editors) (Eli Lilly & Company and the NationalCenter for Advancing Translational Sciences, Bethesda (Md.); 2012)),will be followed.

The lead compounds were fully chemically-modified (meaning nonon-modified riboses remained) using an alternating 2′-O-methyl/2′-Fpattern. The combination of 2′ OMe/2′-F is known to block innate immuneresponse activation (Nair, J. K. et al. MultivalentN-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes andElicits Robust RNAi-Mediated Gene Silencing. Journal of the AmericanChemical Society (2014)). Lack of interferon pathway activation isconfirmed with an in vitro human whole blood cytokine activation assaylooking at IL-1β, IL-1RA, IL-6, IL-8, IL-10, IL-12(p70), IP-10, G-CSF,IFN-γ, MCP-1, MIP-1α, MIP-1β, and TNF-α (Bio-Plex Pro Magnetic CytokineAssay; BioRad Laboratories) and in vivo (after injection in mice)looking at G-CSF, TNF, IL-6, IP-10, KC, and MCP-1 (Cytokine/ChemokineMagnetic Bead Panel; Millipore) (Kumar, V. et al. Shielding of LipidNanoparticles for siRNA Delivery: Impact on Physicochemical Properties,Cytokine Induction, and Efficacy. Molecular therapy. Nucleic acids 3,e210 (2014)).

Without intending to be bound by scientific theory, based on data fromother oligonucleotide chemistries (Wooddell, C. I. et al.Hepatocyte-targeted RNAi Therapeutics for the Treatment of ChronicHepatitis B Virus Infection. Molecular therapy: the journal of theAmerican Society of Gene Therapy 21, 973-985 (2013); Coelho, T. et al.Safety and efficacy of RNAi therapy for transthyretin amyloidosis. TheNew England journal of medicine 369, 819-829 (2013)), the dose limitingtoxicity will most likely be related to liver function. Preliminarystudies determined that up to 50% of the injected dose of the hsiRNAsaccumulated in liver, with delivery being specific to endothelial,kupffer and stellate cells, not hepatocytes (FIG. 4A). With otherphosphorothioate-containing oligonucleotides, slight reversibleelevation of liver enzymes and mild reversible injection side reactionshave been noted as side effects (Frazier, K. S. AntisenseOligonucleotide Therapies: The Promise and the Challenges from aToxicologic Pathologist's Perspective. Toxicologic pathology 43, 78-89(2015)), but usually this liver enzyme elevation is only observed afterlong-term continuous dosing with high dose levels. Because thistreatment is necessarily short-term (just one or two injections over aperiod of one to two months) and does not target hepatocytes, livertoxicity may not be an issue. Nonetheless, these concerns will bestudied in detail.

Development of any therapeutic targeting pregnant women has additionalsafety considerations. A major concern is potential transfer of hsiRNAsto the fetus and any possible toxicity this might cause. In preliminarystudies no detectable oligonucleotide transfer to the fetus was observedusing fluorescent microscopy (FIG. 6A), or using a highly sensitive PNA(Peptide Nucleic Acid)-based quantitative assay (FIG. 4B). Nor were anyeffects on fetal growth, number of miscarriages, placental histology orother teratogenic effects observed.

1.5 Assay Systems in Place to Evaluate Lead Compounds.

The assays and models developed so far are as follows.

Fluorescence Microscopy Evaluation of In Situ Tissue Distribution

hsiRNA variants with a Cy3 or Cy5.5 (lower auto-fluorescence) dyeattached through a non-degradable linker to the 5′ end of sense(passenger) strand were synthesized. This compound was biologicallystable with no detectable Cy3 cleavage within 24 hours. The fluorescentsense strand hybridized to its complementary guide strand (thus forminga double-stranded hsiRNA) was administrated to animals andoligonucleotide distribution patterns were examined in 4 μm tissuesections also stained with DAPI or/and cell type selective antibodies.Parallel sections could be stained with standard histology markersenabling detailed histology mapping. Because hsiRNAs are already heavilyhydrophobically modified, dye addition has little effect on overallhydrophobicity and therefore minimal impact on oligonucleotidedistribution. This assay allowed rapid evaluation of tissue andcell-type distribution and was complemented by a PNA-based quantitativeassay for direct guide strand detection.

PNA Hybridization for Quantitative Guide Strand Detection in TissueLysates

To enable direct quantification of intact guide stand in tissues, anovel assay was developed and implemented wherein the guide strand washybridized to a fully complementary Cy3-labeled PNA (peptide nucleicacid) oligonucleotide, and the corresponding duplex was separated fromexcess single stranded PNA by HPLC (FIG. 2). Since PNA is non-chargedand has extremely tight binding to the guide strand, it out-competesboth the hsiRNA sense strand and any endogenous target sequences.Fluorescence detection of the Cy3-PNA:guide hybrid provided a directmeasure of guide strand abundance in tissue lysates. In conjunction withan HPLC auto injector, this assay enabled guide strand quantification inhundreds of samples overnight. The assay was also highly sensitive, witha limit of detection less than 10 fmole/gram, and hybrids containingfull-length, partially degraded, 5′-phosphorylated and5′-dephosphorylated guide strand can all be quantified as separate peaksor shoulders in the HPLC trace. Because this assay could detect bothlabeled and unlabeled compounds, it can be directly transitioned tofuture CRO's for clinical sample analysis.

QuantiGene® (Affymetrix) Assay for Direct Detection of Flt1 mRNAVariants in Cells and Tissues

QuantiGene® is a highly sensitive 96-well based assay in which mRNA isdirectly detected through signal amplification directly from tissueand/or cell lysates. By linking this direct detection assay to a 192well automatic TissueLyser, a high-throughput version was developedwhich enabled processing of dozens of samples per animal. Thus,quantitative data on expression of targeted and housekeeping genes wasgenerated in many animals at once. In pilot studies, n=8 was sufficientto detect 40% modulation of sFlt1 mRNA isoform expression with 80%confidence. bDNA assays are described in Coles et al. Nucleic Acid Ther.(2015) Nov. 23. PMID: 26595721.

ELISA (#MVR100, R&D Systems) for Detection of sFLT1 Proteins inConditioned Media and Blood

This 96-well based assay required only 10 μL of biological fluid persample. This assay has been optimized over many years for both in vitroand in vivo studies. It is clinically compatible and allows forevaluation of circulating sFLT1 protein levels without animal sacrifice,and will be particularly useful for non-human primate studies.

Normal Mouse Pregnancy Model

The sFlt1-i13 variants are expressed during mouse pregnancy with i13levels exponentially increasing from days 14-19. Perfect homologybetween the sFLT1-i13-2283 compound and the i13 mouse variant allows thestudy both of efficacy and of safety in this simple rodent model.

Preeclampsia Models

Reduced Uterine Perfusion Pressure (RUPP) model of placental ischemiaand hypoxia model of preeclampsia is used as described further below.

Baboon Wild-Type Pregnancy Model

The sFlt1-i15a variant is not expressed in rodents during pregnancy,thus overall combination efficacy and safety will be evaluated inwild-type pregnant baboons using ELISA, a non-invasive assay as readoutof efficacy.

Preliminary Data

A simple and cost-effective PE therapeutic using RNAi to limit excessplacental expression of sFLT1 proteins was developed. For this to work,the following objectives were achieved: (1) appropriate siRNA targetingsites in sFlt1 mRNAs were identified; (2) whether RNA silencing waspossible in the placenta using generalized (i.e., intravenous orsubcutaneous) delivery was determined; and (3) novel siRNA chemistrieswere developed that would enable preferential delivery to placentaltrophoblasts, the cell type responsible for excess sFLT1 production.

Using tissue-specific RNA-Seq data available from the Human ProteinAtlas (See proteinatlas [dot] org) and PAS-Seq data from multiple normaland PE human placentas (FIGS. 1B-C), it was determined that, while thefull length (fl) isoform predominates in all tissues in non-pregnantadult humans, placental expression is dominated by three truncatedisoforms, sFlt1-i13-short, sFlt1-i13-long and sFlt1-i15a, generated bypolyadenylation within introns 13 and 15, respectively. Targeting theintronic regions with hsiRNAs enabled selective silencing of truncatedisoforms without interfering with fl-Flt1 mRNA abundance.

A novel type of siRNA chemistry was developed that enabled efficientdelivery to endothelial cells and demonstrated selective trafficking tothe labyrinth region of the placenta (i.e., to trophoblasts, the celltype responsible for sFLT1 expression). Without any additionalformulation, up to 12% of the injected dose accumulated in the placentawith no detectable fetal transfer. This technology is the firstdemonstration of selective labyrinth targeting by any ONT, enablingsilencing of sFLT1 protein at it major site of expression (FIG. 6A).

Over 50 siRNA variants were designed and screened (See FIG. 13).Hyper-functional, fully chemically-modified hsiRNAs were identified thatselectively targeted the i13 and i15 isoforms without interfering withfl-FLT1 expression (FIG. 3). Using these hsiRNAs, efficient silencing ofi13 and i15 was demonstrated in primary human trophoblasts with noactive formulation (i.e., chemically unassisted internalization/uptakewithout the need for lipid) (FIG. 2B). A combination of sFLT1-i13-2283and sFLT-i15a-2519 hsiRNAs was selected as the lead candidate fortreatment of PE (FIG. 3).

It was determined that in-tissue compound concentrations in pregnantmice could reach 100 μg/gram with a single subcutaneous (SC) orintravenous (IV) injection, producing more than 50-80% reduction insFlt1-i13 mRNA (FIGS. 6 and 4, respectively). Without intending to bebound by scientific theory, with this level of delivery, silencing isexpected to persist for weeks in humans, and thus a limited number ofinjections to be necessary. Indeed, just one SC injection could besufficient to silence sFLT1 for several weeks, resulting in significantPE pregnancy extension, possibly even to full-term.

Example 2. Hydrophobically Modified siRNAs (hsiRNA): FullyChemically-Modified siRNA/Antisense Hybrids

A panel of chemistries and formulations were considered as potentialapproaches for placental delivery. These included LNA antisense, LNPs,chol-conjugates/DPC GalNacs and hsiRNA. hsiRNAs by far exceeded otherchemistries in placental delivery (discussed further infra) and wereselected for further investigation. The efficiency of hsiRNA uptake inprimary trophoblasts was evaluated. Efficient uptake by all cells uponaddition of Cy3-labeled compound to the media was observed (FIG. 2B).The hsiRNAs are asymmetric compounds, with a short duplex region (15base-pairs) and single-stranded fully phosphorothioated tail, where allbases are fully modified using alternating 2′-F/2′-O-methyl pattern(providing stabilization and avoidance of PKR response), and the 3′ endof the passenger strand is conjugated to TEG-Cholesterol (FIG. 3A). Thecholesterol enabled quick membrane association, while thesingle-stranded phosphorothioated tail was essential for cellularinternalization by a mechanism similar to that used by conventionalantisense oligonucleotides (D. M. Navaroli, J. C., L. Pandarinathan, K.Fogarty, C., Standley, L. L., K. Bellve, M. Prot, A. Khvorova and &Corvera, S. Self-delivering therapeutic siRNA internalization through adistinct class of early endosomes. PNAS, under review, secondresubmission (2015)). Addition of Cy3-labeled hsiRNA to any culturedcell type shows quick and efficient internalization through an EE1related part of the endocytosis pathway. A previous version of thistechnology (Byrne, M. et al. Novel Hydrophobically Modified AsymmetricRNAi Compounds (sd-rxRNA) Demonstrate Robust Efficacy in the Eye.Journal of ocular pharmacology and therapeutics: the official journal ofthe Association for Ocular Pharmacology and Therapeutics (2013)), whereonly 50% of bases are 2′F/2′-O-methyl modified, is in Phase II clinicaltrials for dermal fibrosis.

A chemical modification pattern that does not interfere with primaryRISC entry was developed. A wide range of chemical variations weregenerated and an alternating 2′F/2′-O-methyl pattern was identified thatoptimally configures the guide strand to adopt a geometry that closelymimics that of an individual strand in an A-form RNA duplex. The A-formRNA duplex is recognized by the RISC complex and supports properpositioning of the target mRNA within the cleavage site (Ameres, S. L.,Martinez, J. & Schroeder, R. Molecular basis for target RNA recognitionand cleavage by human RISC. Cell 130, 101-112 (2007); Schirle, N. T.,Sheu-Gruttadauria, J. & MacRae, I. J. Gene regulation. Structural basisfor microRNA targeting. Science 346, 608-613 (2014)). By starting thealternating pattern with a 5′-phosphorylated 2′-O-methyl ribose (a 5′phosphate is necessary for PIWI domain interaction), the 2′Fmodifications were placed in even numbered positions 2-14. Positions 2and 14 were previously shown to be intolerant of bulkier 2′-ribosemodifications (Jackson, A. L. et al. Position-specific chemicalmodification of siRNAs reduces “off-target” transcript silencing. Rna12, 1197-1205 (2006); Kenski, D. M. et al. siRNA-optimized Modificationsfor Enhanced In Vivo Activity. Molecular therapy. Nucleic acids 1, e5(2012)).

These fully chemically stabilized compounds were at least as or moreeffective as naked siRNA in RISC entry and represent the first completechemical modification pattern with no negative impact on RISC function.This discovery was transformative for the PE project, as completechemical stabilization is absolutely essential for tissue accumulationupon systemic administration. FIG. 8 shows that no full-length compoundcould be detected in mouse placentas 24 hours post administration of aversion wherein 40% of the riboses were still 2′-OH (P0 chemistry). Incomparison, both fully 2′-F/2′-O-methyl modified versions (P1 and P2chemistries) accumulated to above therapeutically efficacious levels(FIG. 8). Another benefit of non-RNA containing siRNAs is ease ofmanufacturing—their DNA-like chemistry with no necessity for orthogonalribose protection shortens de-protection procedures and increasescoupling efficiencies. Finally, complete elimination of all 2′-OH groupshelps with avoidance of the innate immune response, which relies mainlyon 2′-OH interactions (Alexopoulou, L., Holt, A. C., Medzhitov, R. &Flavell, R. A. Recognition of double-stranded RNA and activation ofNF-kappaB by Toll-like receptor 3. Nature 413, 732-738 (2001); Choe, J.,Kelker, M. S. & Wilson, I. A. Crystal structure of human toll-likereceptor 3 (TLR3) ectodomain. Science 309, 581-585 (2005)).

FM-hsiRNAs were determined to be more potent in passive uptake thannon-fully modified hsiRNAs in primary trophoblasts (FIG. 31). Fullmetabolic stabilization was determined to be essential for systemicdelivery following intravenous administration (FIG. 32). FM-hsiRNAs werealso determined to be essential for systemic delivery followingsubcutaneous administration (FIG. 33).

A PNA-based assay was developed to quantitate guide strand distributionin vivo (FIG. 34). Using this assay, robust delivery and efficacy toliver and kidney tissues were observed in vivo after administration ofFM-hsiRNAs (FIGS. 35A-35F).

In vivo stability in liver tissues after IV or SC administration wasassayed at 120 hours post-IV and post-SC administration (FIGS. 18A-B).hsiRNA levels were assayed in vivo at two hours, 24 hours and 120 hourspost-IV administration (FIG. 19).

Example 3. hsiRNAs Enabled Selective Delivery to Placental LabyrinthTrophoblasts with No Detectable Fetal Transfer

To evaluate hsiRNA distribution in vivo, normal pregnant mice (day 15)were injected with Cy3-labeled sFlt-i13-2283 hsiRNA and distributionexamined at by two independent assays. Gross tissue fluorescencemicroscopy revealed that most of the oligonucleotides accumulated tothree tissues: liver endothelium, kidney endothelium and placentallabyrinth (FIG. 4). Without intending to be bound by scientific theory,this distribution profile was most likely defined by a combination ofblood flow/filtration rate and the cholesterol receptor concentration oncell surfaces. Using the novel FDA-compliant PNA-hybridization assaydescribed above, it was demonstrated that overall drug concentration inplacenta exceeded efficacious levels (approximately 100 ng/gram) byorders of magnitude upon a single 10 mg/kg injection (FIG. 8). Thislevel of tissue delivery was roughly the same for IV and SCadministration, with approximately 50%, 10% and 12% of the compounddistributing to liver, kidney and placenta, respectively, 24 hourspost-injection (FIG. 4). Interestingly, only half of this was clearedfrom the liver (slightly more in kidney) after five days, indicatingthat a single administration might be sufficient to induce long-termsilencing.

FIG. 7A shows oligonucleotide distribution in a 4 μM sagittal slice cutthrough a fetus and its attached placenta. It was amazing and highlysatisfying to observe efficient delivery to the placental labyrinth withessentially no detectable oligonucleotide transfer to the fetus,including the fetal liver. These data were independently confirmed bythe PNA assay which could detect no hsiRNAs in fetal liver (FIG. 4B)(sensitivity of the assay was approximately 10 fmole/gram). FIG. 5depicts histological analysis of the placenta and confirmed specificdelivery of hsiRNAs to placental labyrinth trophoblasts, the major celltype responsible for sFLT1 expression. Remarkably, almost no Cy3 wasdetectable in other layers (e.g., junctional and decidua), furthersupporting the specificity of this novel chemical modification patternfor delivery to the labyrinth trophoblasts.

In addition to comparing the impact of full 2′-F/2′-O-methylmodification on PK (pharmacokinetics), the phosphorothioate (PS) contentwas slightly altered. While the P1 chemistry had PS linkages at the3′-ends of both strands (for a total of 8), the P2 chemistryincorporated another two PS's at the 5′ end of each strand (for a totalof 12). Terminal PS linkages provided a defense against exonucleases,and so are essential for long-term stability in extremely aggressivenuclease environments. Overall, these two chemistries were comparable inlevels of oligonucleotides delivery at 24 hours (FIG. 8), but might havedifferent degradation profiles after long term tissue exposure,affecting duration of the silencing effect. They also have slightlydifferent liver: placenta distribution ratios, which might also besomewhat affected by the route of administration (FIG. 8).

3.1. Selection and Identification of Lead Candidate: i13/i15 Mix andEfficacy in Primary Trophoblasts

The i13 and i15 Flt1 mRNA isoforms contained 435 and 567 uniquenucleotides, respectively, not present in fl-Flt1 mRNA. Unfortunately,the majority of this sequence space was dominated by homo-polymericrepeats and regions of high GC content, neither of which are targetableby RNAi. Undeterred, a panel of more than 50 hsiRNAs was designedagainst any feasible targetable sequence using standard siRNA designparameters (Birmingham, A. et al. A protocol for designing siRNAs withhigh functionality and specificity. Nature protocols 2, 2068-2078(2007)) including assessment of GC content, specificity and low seedcompliment frequency (Anderson, E. M. et al. Experimental validation ofthe importance of seed complement frequency to siRNA specificity. Rna14, 853-861 (2008)), elimination of sequences containing miRNA seeds,and examination of thermodynamic bias (Khvorova, A., Reynolds, A. &Jayasena, S. D. Functional siRNAs and miRNAs exhibit strand bias. Cell115, 209-216 (2003); Schwarz, D. S. et al. Asymmetry in the assembly ofthe RNAi enzyme complex. Cell 115, 199-208 (2003)). FIG. 6B shows thetargeting positions of hsiRNAs identified to be highly functional.

In the design criteria, targeting sites with perfect homology in otherprimates were favored to simplify both formal toxicology and efficacystudies in non-human primates and the baboon PE model described below.The mouse expresses only an i13 variant. Luckily, the most efficacioushsiRNA, sFLT1-i13-2283, happened to have perfect complementarity to themouse i13 isoform, enabling direct in vivo efficacy and toxicityevaluation of this compound in both normal and PE mouse pregnancymodels. FIG. 6C shows a table with targeting sites and IC50 values ofthe best compounds identified to efficiently silence the i13 and i15isoforms. IC50 values for efficacious compounds ranged between 40-100 nMin both HeLa cells and primary human trophoblasts.

FIG. 3C shows an example of the dose response of sFLT1-i13-2283 inprimary human trophoblasts used for IC50 value calculation. It isimportant to emphasize that silencing with hsiRNAs was achieved uponaddition of non-formulated compound to the trophoblast media. The levelof mRNA knockdown was determined at 72 hours using the above-describedQuantiGene assay. To control for any potential non-specific effects, i13or i15 levels were always normalized to a housekeeping gene. ANon-Targeting-Control (NTC) of identical chemistry was used in allexperiments to control for chemical class effects. The levels of fulllength Flt1 mRNA were not affected (FIG. 3D). To evaluate silencing atthe protein level, sFLT1 concentration in conditioned medium wasmeasured using ELISA (Quantikine® FLT1, MVR100, R&D Systems) (FIG. 3B).

To move forward, two hsiRNA pairs were selected: sFLT1-i13-2283 (5′CTCTCGGATCTCCAAATTTA 3′) (SEQ ID NO:1)/sFLT-i115a-2519 (5′CATCATAGCTACCATTTATT 3′) (SEQ ID NO:2) and sFLT1-i13-2318 (5′ATTGTACCACACAAAGTAAT 3′) (SEQ ID NO:3)/sFLT-i15a-2585 (5′GAGCCAAGACAATCATAACA 3) (SEQ ID NO:4) (FIG. 3C). The first pair was thelead drug candidate and was used in all studies. The second pair was abackup. While sequence-specific toxicity will unlikely be an issue, abackup compound combination that was readily available in case of anysequence-dependent toxicity appeared was desired. In summary, afunctional hydrophobically modified siRNAs that selectively targetedsFlt1-i13 and sFlt1-i15a isoforms was identified. Efficientinternalization and silencing of the corresponding targets in primaryhuman trophoblasts was determined at both the mRNA and protein levels.

In vitro validation of sFLT1_2283/2519 (sFLT1-mix; 151005 or 151111)showed dose responses for targeting sFLT1 i13 and sFLT1 e15a (FIGS.27A-D and 28A-D).

3.2. sFLT1-i13 Variant Silencing In Vivo Upon Systemic Administration toNormal Pregnant Mice

FIG. 7B shows pilot data demonstrating efficient silencing of sFlt1mRNAs in kidney, liver and placenta subsequent to two 20 mg/kg IVinjections. 12 pregnant mice were dosed with 20 mg/kg sFLT-13-2283compound IV daily for two days, and the level of sFlt1-i13 expression(normalized to both a housekeeping gene and fl-FLT1) was determined 5days later in maternal liver, kidney and placenta, as well as in fetallivers. Statistically significant silencing (50-60%) was achieved in allmaternal tissues and placenta, while levels of sFLT1 expression in fetalliver were not changed. The lack of silencing in fetal liver wasconsistent with the lack of detectable oligonucleotide in this tissue(FIG. 4B). Placental hsiRNA concentration was around 20-40 μg/gram. Thisfar exceeded the concentration necessary for productive silencing, whichis usually observed with compound concentrations as low as 100 ng/gram.The dose used was thus clearly much higher than necessary. The doseresponse and duration of effect were studied in detail, and the NOEALand MTD dose levels were defined for this compound. In addition,maternal weight, placental weight and number and weight of fetuses weremonitored, and it was determined that each of them was unaffected at 10mg/kg injection and slightly affected (average of 6 vs. 7 fetuses/dam)at 20 mg/kg injection levels with no other observable changes. It wasreassuring that, even at this excessively high dose, no hsiRNA transferto the fetus was observed. Based on the drug concentrations achieved inthe placenta, the effective dose should be at least an order ofmagnitude lower.

Histological evaluation of hsiRNA distribution in mouse placentaltissues was performed (FIG. 15). Efficient silencing of sFLT1 by hsiRNAwas observed in liver, kidney and placental tissues of pregnant CD1 mice(FIGS. 16A-16E, FIGS. 17A-D).

Soluble sFLT1 protein modulation was detected in the serum of pregnantmice after a single IV injection (10 mg/kg) of sFLT1_2283/2519 at days14, 15, 17 and 19 (FIG. 29). There were no observable negative effects,nor were there any observable deviations in weight, ALT values, ASTvalues or number of pups per pregnant mouse.

In summary, these data indicate that a novel siRNA chemistry has beendeveloped that has enabled efficient delivery to placental trophoblasts,the primary site of sFLT1 overexpression during PE, and has allowedpotent silencing of circulating sFLT1 upon systemic administration.

Example 4. Chemistry and Optimal Dosing, Pilot PK/PD, and Duration ofEffect of 2283 in a Wild-Type Pregnant Mouse Model

4.1. Chemistry Optimization

Although the modification patterns showed efficient delivery tocytotrophoblasts within the placental labyrinth (FIGS. 4, 5, 7 and 8),two additional chemistry issues will be addressed: 1) furtheroptimization of phosphorothioate (PS) content and 2) furtherstabilization of the 5′-terminal phosphate of the guide strand.

Phosphorothioate (PS) Content

While PS linkages generally confer greater in vivo siRNA stability,extensive phosphorothioation can induce greater class specific toxicity(although only upon prolonged administration at high dose) (Frazier, K.S. Antisense Oligonucleotide Therapies: The Promise and the Challengesfrom a Toxicologic Pathologist's Perspective. Toxicologic pathology 43,78-89 (2015)). Although the P2 chemistry (FIG. 8) is expected to be muchmore stable over long time periods (i.e., weeks, as has been shown forGalNac conjugates) (Nair, J. K. et al. MultivalentN-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes andElicits Robust RNAi-Mediated Gene Silencing. Journal of the AmericanChemical Society (2014)) than P1, pilot studies showed no significantdifference between sFLT1-2283-P2 and sFLT1-2283-P1 with regard toplacental accumulation at 24 hours (FIG. 8). It was therefore suspectedthat the optimal PS content (enough to maintain stability and efficacybut minimize toxicity) lies somewhere between P1 and P2. Synthesis ofseveral additional variants is planned. The concentrations offull-length species in placentas 24 hours and 5 days postadministration, and relative distributions at the injection site and inliver, kidney and placenta will be determined, and the Maximum ToleratedDose (MTD) upon single administration will be established. Based onthese data, a PS pattern will be selected and used for all subsequentstudies.

5′-Terminal Phosphate Stabilization

The second issue is how best to optimize 5′-terminal phosphatestabilization of the guide strand, which strand is necessary for RISCloading. The PNA-based hybridization assay that was used allowedefficient separation of phosphorylated and dephosphorylated guidestrands, has revealed that more than 70% of sFLT1-2283-P2 guide stranddetectable in the liver 5 days post injection is dephosphorylated.Without intending to be bound by scientific theory, it is likely thatintroduction of metabolically stable 5′-(E)-vinylphosphonate (5′-VP)(Lima, W. F. et al. Single-stranded siRNAs activate RNAi in animals.Cell 150, 883-894 (2012)) or 5′(R)Me-P, both chemistries withconformation and sterioelectronic properties similar to the naturalphosphate, instead of 5′phosphate might potentially reduce the effectivedose (and thus drug cost and any class limiting toxicity) by more thanthree-fold. sFLT1-2283 will be synthesized with a 5′-VP and 5′(R)Me-P onthe guide strand (routinely done at Dr Khvorova lab) and evaluate itsimpact on oligonucleotide efficacy, placental delivery and safety. Thetoxicity profiles of 2′-O-methyl, 2′-fluoro, cholesterol and PS are wellunderstood. Completion of this analysis should finalize chemicalconfiguration (PX) of the pre-clinical candidate oligonucleotides forthe treatment of PE.

4.2. Midscale Oligonucleotide Synthesis

Using the chemistry selected in Aim 1.1, sFLT-i13-2283-PX will besynthesized, HPLC purified and Quality Controlled (QC'ed) by massspectrometry. For all in vivo studies, compounds will be desalted,complexed with sodium counter-ion, and checked to ensure that endotoxinlevels are acceptable. The oligonucleotides were previously synthesizedusing Expedite and Mermaid 8 systems and purified via Agilent mid-scaleHPLC. The current synthesis capacity is 40 μmol of compound/week,resulting in 0.2 gram after HPLC purification. This range is sufficientto perform all planned mouse studies (as reference, a 10 mg/kg dosecorresponds to 0.4 mg/mouse/injection for 40 gram pregnant animals). Inanticipation of needing increased synthesis capacity for non-humanprimate studies, a midscale OligoPilot synthesizer and high resolutionLC-MS will be utilized, which will also decrease costs ofoligonucleotide synthesis.

4.3. Pilot Pharmacokinetics (PK), Pharmacodynamics (PD), No ObservedAdverse Effect Level (NOAEL), and Maximum Tolerated Dose (MTD)Measurements for sFLT-i13-2283

All animal studies will be performed under IACUC protocols. In design ofall experiments, the standards recommended by Landis (Landis, S. C. etal. A call for transparent reporting to optimize the predictive value ofpreclinical research. Nature 490, 187-191 (2012)), including propergroup randomization, blinding and study powering, will be followed. Thedevelopment of PNA based chromatographic assay enables one the abilityto quantitatively evaluate oligonucleotide distribution and define pilotPK. For pilot PK studies, n-3 is sufficient. For efficacy and durationof effect studies where the readout is reduction in sFLT1 variants mRNAand protein levels, n-8 is minimally necessary to power the study fordetection of 40% modulation with 80% confidence.

For the pilot PK analysis, blood concentration dynamics will beexplored. 20 μL blood samples will be collected at 5, 30, and 60 min, 4,12, 24, 48, 72 and 96 hours post-injection. Short time points will beobtained by jugular vein catheterization, eliminating IACUC concernsregarding repetitive blood draws over short time periods and minimizingthe number of animals required to obtain tight data. Based on previousPK studies with related compounds, it is expected that biphasicclearance kinetics will be observed, with the fast phase complete by 1and 4-6 hours for IV and SC administration, respectively. Based on thepilot studies showing that >50% of the originally delivered dosepersisted in tissues even 5 days post injection, it might take a monthfor complete drug clearance, but a week-long study would be sufficientto generate pilot data allowing estimation of the clearance profile.

In addition, NOAEL and MTD will be established for the lead compound. Sofar, administration of 10 mg/kg and 20 mg/kg sFLT1-2283-P2 to pregnantmice had had no observable negative impact other than a slight decreasein fetus number at 40 mg/kg. As approximately 50% of the injected dose(FIG. 4B) went to liver endothelium and kupffer cells, it is reasonableto expect modulation of liver enzymes with ALT and AST used as astandard readout. ALT/AST levels will be measured (assays in place aspart of the animal care core) at different sFLT1-2283-PX concentrationsand at different times after injection. This assay can be easilycombined with dose response efficacy studies as it is non-invasive andrequires only 10 μL of blood.

MTD will be established in both pregnant and non-pregnant mice. Innon-pregnant mice, the pilot studies indicated that the sFLT1-2283-P2MTD range was expected to be higher than 100 mg/kg. In pregnant mice,however, the NOAEL dose level might be lower. It is expected that theNOAEL dose will be much higher than the expected efficacious dose of 1mg/kg based on drug levels achievable in the placenta. 1 mg/kg is inline with concentrations used for the ALN-TTR2 compound in phase IIclinical trials.

Oligonucleotide-related toxicity may be due to target-specific effects(sFlt1 related) or target-independent effects (related to the siRNAsequence or formulation). Target specific effects will be evaluated inmice administered the lead compound during the 3rd trimester byassessing fetal number, weight and growth as well as miscarriagefrequency. Placental histology will assess trophoblast and vasculardensity. Uterine artery and umbilical artery blood flows will beevaluated using non-invasive Doppler studies (Zhang, J. H. & Croy, B. A.Using Ultrasonography to Define Fetal-Maternal Relationships: Movingfrom Humans to Mice. Comparative Med 59, 527-533 (2009)). In addition,mice treated with the NOAEL and MTD dose will be allowed to deliver andthe pups followed for two generations to evaluate potential impact onoverall health and reproductive function.

For PD studies, a dose response study will be performed with dosesranging from 0.5 to 10 mg/kg. sFLT1 protein levels will be measured withELISA (blood kinetics) and sFlt1 and fl-Flt1 mRNA levels will bemeasured with QuantiGene in placenta, liver and kidney. Preliminarystudies showed that a single administration of sFLT1-i13-2283-P2 wassufficient to induce silencing of sFLT1 in all targeted tissues (FIG.7B) with no effect in embryos livers. In vivo validation ofsFLT1_2283/2519 is shown in FIG. 7C. The effect duration will beanalyzed at both the highest NOAEL and lowest efficacious dose.Unfortunately, mice might not be an optimal model for these studies, assFLT1 is barely expressed in non-pregnant mice and pregnant mice do notallow for PD evaluation at times longer than 2 weeks. To evaluate thepotential for longer duration studies, hsiRNAs sharing exactly the samechemistry that target TEK tyrosine kinase (Tie2) mRNA, an endothelialspecific target, will be utilized. Tie2 silencing will be examined inliver endothelium 1, 2, 3, 4 weeks after a single administration. Ifsilencing does not persist for more than 3 weeks, multiple dosing willbe explored to address the issue. While the PK/PD behavior of differentsequences is not identical, it is similar (H. Younis et al. in AComprehensive Guide to Toxicology in Preclinical Drug Development. (ed.A. S. Faqi) 647-664 (Academic Press, 2013)), and effect duration datawith Tie2 compounds might be informative for design of NHP efficacystudies.

This study will result in chemical configuration finalization andestablish the dose response, pilot PK, NOAEL and MTD for sFLT1-2283 innon-pregnant and normal pregnant mice. This information will be criticalfor further planning of IND-enabling toxicology studies.

4.4 Pilot Safety and Toxicity of i13/i15 Combination.

The current pre-clinical candidate is defined as a mixture of hsiRNAstargeting all i13 and i15 variants. Both compounds may be re-synthesizedbased on the most optimal chemical composition identified above. If thecompounds are re-synthesized, it will be essential to demonstrate thatthe i13/i15 mixture has a similar safety profile to targeting the i13variant alone. Experiments to determine the NOAEL and MTD for themixture of compounds will be repeated using AST/ALT readouts as measuresof toxicity. In addition, similarity in PK behavior will be confirmedfor i15 and i13 targeting compounds using i13 and i15 targetingPNA-based probes. It is impossible to evaluate PD of i15 in the mousemodel as i15 variants are not expressed there. Without intending to bebound by scientific theory, it is believed that having similar in vitro(i.e., trophoblast) efficacy in combination with similar PK profileswill be sufficient to predict similar efficacy. This will be confirmedin a baboon pregnancy model where both i13 and i15 variants areexpressed.

4.5 Quantitatively Assess hsiRNA Transfer (if any) to the Fetus

The major safety concern in developing any therapeutic for treatment ofpregnant women is fetal safety and potential negative impact on embryodevelopment. In the preliminary data presented herein, no detectabletransfer of sFLT1-2283-P2 to the fetus was observed (FIGS. 6A and 4B)either by fluorescent microscopy and PNA hybridization. Nor was anychange in fetal sFLT1 expression observed in a drug-treated group ofanimals. At doses up to 20 mg/kg, no impact was observed on fetus weightor numbers, and no impact on overall placenta histology was observed. Aslight decrease in fetus numbers (from average of 7 to 6) was observedat a maximal dose of 40 mg/kg. In spite of this encouraging data whichmakes this project so promising, it is important to quantitativelyanalyze any potential drug transfer and impact on the placenta and fetusin detail. To do so, a PNA hybridization assay will be used to detectoligonucleotides in different embryo tissues. As mentioned above, thesensitivity of the assay is approximately 10 fmole/gram, lower thanbiologically efficacious concentrations. The level of potentialoligonucleotide transfer will be measured after administration ofdifferent concentrations of oligonucleotide up to MTD. These experimentswill also be repeated using PE models, as the integrity and health ofplacenta might impact the barrier ability.

One concern particular to siRNA therapeutics is the potential for even asmall amount of transferred oligonucleotide to interfere with miRNAhomeostasis. miRNA profiles change rapidly during embryo development andare essential for proper execution of the development program.Previously, altered miRNA profiles were seen only after months longvirus-based overexpression of a particular siRNA (Grimm, D. et al.Fatality in mice due to oversaturation of cellular microRNA/shorthairpin RNA pathways. Nature 441, 537-541 (2006)). Cell-specific miRNAsignatures are highly dynamic during embryonic development, thuspotentially more sensitive than adult tissues to external RISC loadingsubstrates. To examine this possibility, RNA will be purified from fullfetuses, fetal brain and fetal liver from untreated and MTD-dosedpregnant mice and perform small RNA-Seq. This will enable: (1)evaluation of the number of reads corresponding to the hsiRNAs in thesetissues and (2) evaluation of impact (if any) on endogenous miRNAprofiles.

Example 5. Additional PE Models

5.1 Demonstration of Efficacy of sFLT1 Oligonucleotide in Two Models ofPreeclampsia

While reduction in sFLT1 levels supports mechanistic efficacy of thelead, it would be desirable to demonstrate that sLFT1 reduction has animpact on the “preeclampsia-like” phenotype. While there aredescriptions of several animal models of preeclampsia in the literature,no single model recapitulates all aspects of the clinical syndrome, andnone of them accurately models progression of the disease frompreeclampsia to more serious complications such as HELLP syndrome oreclampsia (Aubuchon, M., Schulz, L. C. & Schust, D. J. Preeclampsia:animal models for a human cure. Proceedings of the National Academy ofSciences of the United States of America 108, 1197-1198 (2011)). Thus,there is as yet no perfect model for the human disease. Because mice andrats have relatively shallow placentation, rodent models are not optimalfor studying the upstream causes of poor trophoblast invasion. On theother hand, this feature makes them ideal for evaluating the downstreampathophysiology and etiology of the maternal response to shallowplacentation, an established driver of human preeclampsia (Powe, C. E.,Levine, R. J. & Karumanchi, S. A. Preeclampsia, a disease of thematernal endothelium: the role of anti-angiogenic factors andimplications for later cardiovascular disease. Circulation 123,2856-2869 (2011)). Since the overall goal of this invention is toknockdown sFlt1 in human pregnancies, mouse models (uterine ischemia andwhole animal ischemia) that have been reported to have elevatedcirculating sFlt1 were chosen. Relative hypoxia and ischemia have bothbeen reported to induce sFlt1 production in human placental cultures invitro and in various animal models (Nagamatsu, T. et al.Cytotrophoblasts up-regulate soluble fms-like tyrosine kinase-1expression under reduced oxygen: an implication for the placentalvascular development and the pathophysiology of preeclampsia.Endocrinology 145, 4838-4845 (2004); Makris, A. et al. Uteroplacentalischemia results in proteinuric hypertension and elevated sFLT-1. Kidneyinternational 71, 977-984 (2007); Gilbert, J. S., Babcock, S. A. &Granger, J. P. Hypertension produced by reduced uterine perfusion inpregnant rats is associated with increased soluble fms-like tyrosinekinase-1 expression. Hypertension 50, 1142-1147 (2007)).

5.2 Telemetry Surgery

Female CD1 pregnant mice weighing 20-25 grams will be purchased fromCharles River Laboratories. The advent of miniaturized, surgicallyimplantable radiotelemetry probes suitable for chronic hemodynamicmonitoring of small laboratory animals has significantly advancedphysiologic research and is important in preeclampsia research as thetechnique allows one to measure blood pressure throughout gestation(Burke, S. D. et al. Spiral arterial remodeling is not essential fornormal blood pressure regulation in pregnant mice. Hypertension 55,729-737 (2010)). Briefly, female CD-1 mice weighing over 20 g will beanesthetized using inhaled isoflurane. A 1 cm midline incision is madeon the ventral surface from the sternal notch rostrally. The left commoncarotid artery is isolated and occluded using sutures. The artery isincised with a 26 gauge needle and the telemetry catheter (DSI, St.Paul, Mich.) is advanced into the artery; the tip is placed just withinthe arch of the aorta. The catheter is sutured into place, permanentlyoccluding the artery. The body of the transmitter is placed into asubcutaneous pocket on the right flank of the mouse. The wound issutured and the mouse is monitored until recovered. Animals will beprovided with analgesia for 48 hours post-operative and monitored for 1week. Two weeks after recovery, animals will be mated with male studmice of the same background to study pregnancy phenotypes as describedbelow.

5.3 Reduced Uterine Perfusion Pressure (RUPP) Model of PlacentalIschemia

Theo RUPP model of placental ischemia in pregnant CD1 mice that waspioneered by Barbara Alexander Laboratory at the University ofMississippi will be used (Intapad, S. et al. Reduced uterine perfusionpressure induces hypertension in the pregnant mouse. American journal ofphysiology. Regulatory, integrative and comparative physiology 307,R1353-1357 (2014)). This animal model of preeclampsia involves placementof silver constriction clips on the abdominal aorta and above theuterine arteries to reduce uterine perfusion pressure on gestational day14, creating placental insufficiency. Animals will be given either sFLT1oligonucleotide drug (using dose level and route of administrationdefined supra) or control (PBS or/and NTC) on gestational day 16 viatail vein injection. Blood pressures will be measured continuously usingtelemetry from gestational day 15-19 and animals will be sacrificed ongestation day 19. In addition, urine samples will be collected byplacing the mice in metabolic cages for 12 hours prior to sacrifice.

5.4 Hypoxia Model of Preeclampsia

The hypoxia model that has been pioneered by Surendra Sharma laboratoryat Brown University will also be used. Id. In this model, pregnant CD1mice that have been implanted with a telemeter will be exposed tohypoxia (9.5% oxygen, exposed for 10 days) in a hypobaric chamber fromgestational day 9-19. Animals will be given either therapeutic sFLT1oligonucleotide dissolved in PBS (2 doses) or vehicle (PBS) ongestational day 16 via tail vein injection. Blood pressures willmeasured continuously using telemetry from gestational day 15-19 andanimals will be sacrificed on gestation day 19. Urine samples will becollected as described above.

Preeclampsia Phenotypes

Measurements of blood pressures will be performed in conscious miceusing telemetry probe implanted into the carotid artery (DSL, St. Paul,Minn.). (See pilot studies in FIG. 9A.) Renal tissue will be examinedhistologically for quantification of glomerular endotheliosis asdescribed (Li, Z. et al. Recombinant vascular endothelial growth factor121 attenuates hypertension and improves kidney damage in a rat model ofpreeclampsia. Hypertension 50, 686-692 (2007)). Tissue will be fixed in10% buffered formalin, embedded in paraffin, sectioned, and stained withH&E, PAS, and Masson's trichrome stain. Serum and urinary creatinine(measured by picric acid calorimetry) and urinary protein (measured byELISA) during pregnancy (gestational day 18-19) will be measured toevaluate for proteinuria. In addition, hematocrit and platelet countwill be measured on gestational day 18-19. Peripheral smears will beperformed using whole blood obtained from these rats to look forevidence of hemolysis. Serum AST and ALT will be measured by kinetic UVmethod (Infinity Liquid, Thermo Electron Corp). Plasma levels of sFlt1will be measured by commercially available ELISA (R & D Systems, MN).

Placental/Fetal Studies

Ultrasound Doppler will be used to evaluate uterine and umbilical flowsat gestational day 18-19 as described elsewhere (Khankin, E. V., Hacker,M. R., Zelop, C. M., Karumanchi, S. A. & Rana, S. Intravitalhigh-frequency ultrasonography to evaluate cardiovascular anduteroplacental blood flow in mouse pregnancy. Pregnancy hypertension 2,84-92 (2012)). Mice will be anesthetized using Isoflurane/02 mixtureadministered with a precision vaporizer. Mice will be placed in a supineposition on heated stage of Vevo 2100 Ultrasonography Apparatus (VisualSonics Inc. Toronto, Ontario CA) and a rectal temperature probe will beinserted to monitor core temperature throughout the procedure. Abdominalorgans will be scanned and the uterine artery will be identified. Oncethe position is verified, pulse wave Doppler will be used to visualizeblood flow pattern and measure flow velocity in the uterine artery.Ultrasound evaluation will be done on at least 4 embryos per animal: twoin each hom. Measurements that will be taken include Fetal Heart Rate(FHR), Umbilical Artery Doppler (UA Doppler), Uterine Artery Doppler(Ut. A Doppler) and Abdominal Circumference (AC) (FIG. 9B). Litter sizesand resorptions will be scored when animals are sacrificed atgestational day 19. Birth weights will be recorded for evidence of fetalgrowth restriction. Implantation sites with placentas will be fixed in4% paraformaldehyde and will be examined histopathologically forevidence of abnormal spiral artery remodeling, a feature of abnormalplacentation (Cui, Y. et al. Role of corin in trop hoblast invasion anduterine spiral artery remodelling in pregnancy. Nature 484, 246-250(2012)).

Sample Size and Statistical Comparisons

For each of the studies proposed, approximately 10 animals will be usedper group: control, low dose sFlt1 oligonucleotide and high doseoligonucleotide. Standard statistical analyses will be performed on allthe animal data. Individual values will be collated as means+/−S.E.M.Comparisons among multiple groups will be made by an initial analysis ofvariance, and Student's t-test will be used to evaluate differencesbetween individual groups. Hemodynamic data will be analyzed using 24hour means from individual animals data, which will be compared usingtwo-way repeated measures ANOVA. Where significant differences areindicated (p<0.05), Bonferroni's post-hoc test will be used to evaluatedifferences among individual groups.

5.5 Interpretation, Pitfalls and Alternatives.

Without intending to be bound by scientific theory, it is expected thatthe sFLT1 oligonucleotide therapy will lead to an approximately 50%reduction in circulating sFLT1 levels, which will be associated withimprovement in preeclampsia phenotypes such as resolution ofhypertension and improvement in renal pathology.

It is not expected that there will any adverse consequences to fetalgrowth or placentation. Genetic knock-down studies by Rossant's grouphave suggested that placental sFlt1 is not critical for the maintenanceof pregnancy (Hirashima, M., Lu, Y., Byers, L. & Rossant, J. Trophoblastexpression of fms-like tyrosine kinase 1 is not required for theestablishment of the maternal-fetal interface in the mouse placenta.Proceedings of the National Academy of Sciences of the United States ofAmerica 100, 15637-15642 (2003)). However, since sFlt1 will be knockeddown systemically, it is possible that there may be adverseconsequences, such as decreases in fetal growth related do significantdrop in blood pressures. If greater than a 20 mm drop in blood pressuresis not observed, these efficacy studies will be repeated with a lowerdose of the sFLT1 oligonucleotide. Furthermore, with non-invasiveultrasound Doppler, even subtle changes in blood flow to the fetus canbe recorded. If robust expression of endogenous sFlt1 levels in responseto hypoxia is not observed during the 3^(rd) trimester, the studiescould be repeated in IL-10 deficient mice which have been shown bySharma's group to upregulate sFlt1 during third trimester quitedramatically (Intapad, S. et al. Reduced uterine perfusion pressureinduces hypertension in the pregnant mouse. American journal ofphysiology. Regulatory, integrative and comparative physiology 307,R1353-1357 (2014)). Completion of this aim will evaluate efficacy andsafety of RNAi-based sFLT1 reduction in two models of PE.

Example 6. Evaluation of PK and Efficacy and Safety in Pregnant BaboonsDuring Late Gestation in a Pilot Study

6.1 Rationale

Because mice only express one of the isoforms of sFlt1, it is importantto evaluate the in vivo efficacy of sFlt1 oligonucleotide(s) innon-human primates that express all the sFlt1 isoforms (Makris, A. etal. Uteroplacental ischemia results in proteinuric hypertension andelevated sFLT-1. Kidney international 71, 977-984 (2007); Thomas, C. P.et al. A recently evolved novel trophoblast-enriched secreted form offms-like tyrosine kinase-1 variant is up-regulated in hypoxia andpreeclampsia. The Journal of clinical endocrinology and metabolism 94,2524-2530 (2009)). Baboons have been chosen over other non-humanprimates because of access to well characterized model of preeclampsiathat has been pioneered by Dr. Annemarie Hennessy (Makris, A. et al.Uteroplacental ischemia results in proteinuric hypertension and elevatedsFLT-1. Kidney international 71, 977-984 (2007)).

6.2 Baboon Pregnancy Model

Due to ethical constraints to minimize the number of animal subjects,six pregnant baboons (2 groups×3 animals) will be used. At 20 weeks ofgestation, (equivalent to gestation time at which human PE occurs), allsix animals will undergo telemetry surgery to measure intra-arterialblood pressure as previously described. Id. Briefly, an inguinalincision is made in the skin to expose the profunda femoris, a smallbranch of the main artery supplying the leg, via blunt dissection. Thecatheter component of the telemeter (Data Sciences Ltd, Minnesota, USA)is inserted into a tributary of the femoral artery on the selected sideand placed a set distance into the mid-abdominal aorta and secured tothe vessel. Post-surgical recovery is assessed by scoring a number ofdifferent physical and behavioral signs, and is carried out and recordedon a daily basis for three days prior to the commencement of bloodpressure recordings. For anesthesia, animals will be provided withoxygen via facemask and anesthetized by ketamine infusion, andmetoclopramide to prevent emesis. At 22 weeks of gestation, animals willbe injected intravenously with 4 mg/kg body weight of a single dose ofsFLT1 oligonucleotide in phosphate buffered saline (N=3) or phosphatebuffered saline alone (N=3) under anesthesia.

A baboon PE model was developed (FIG. 30). The baboon PE model will beused to test efficacy (PK) and general safety of administration ofsFLT1-targeting hsiRNAs. A pilot evaluation of single dose injection ofsFLT1-i13/e15a targeting hsiRNAs on sFLT1 blood levels and sFLT1 mRNAlevels in kidney and placental tissues will be performed. A telemeterwill be inserted at day 143. Placental ischemia will be induced andhsiRNA will be injected at day 150. Removal of the telemeter will occurat day 164.

The first baboon was injected on day one using 20 mg/kghsiRNA^(SFLT1m)No observable toxic or adverse effects were noted duringthe first two weeks. Initial ALT/AST levels and cytokine panels werenormal, and blood pressures were normalized. Stabilization of the bloodpressure (BP) was observed after hsiRNA injection (2 weeks study) (FIG.37). Positive dynamics were observed for BP and HR for both awake andsleep conditions. A decrease of BP was observed after a single IV hsiRNAinjection (2 weeks study) (FIG. 38).

Baboons two and three are scheduled for injection one to two monthsafter the first baboon was initially injected.

6.3 Plasma sFlt1 Levels and Other Physiological Parameters

Baseline levels of sFlt1 will be obtained from plasma collected on theday of telemeter insertion and weekly, until delivery at 26 weeks. Urinesamples will also be collected on the same days that blood is drawn.Baseline blood pressure will be continuously recorded via telemetry fromgestational week 21 until delivery. Animals will be monitored usingfetal ultrasounds (HDI 3000 instrument and C7-4 probe, or similar)weekly for determining uterine and umbilical blood flows, fetal health(fetal heart rate) and growth measurements (head circumference,biparietal diameter, abdominal circumference, femur length) untildelivery. At delivery, cord blood will be collected to evaluate fortransfer of sFLT1 oligonucleotide across to the fetus. At delivery,growth assessments will be made by measurements of head and chestcircumference, abdominal girth, crown-to-rump and femur lengths. Thesemeasurements will be compared against those obtained from newborns ofsaline treated animals.

6.4 Interpretation, Pitfalls and Alternatives

In these pilot studies, the goal is not to find the optimal therapeuticdose for treatment of preeclampsia, but to simply confirm that sFLT1oligonucleotide therapy inhibits other isoforms of sFLT1 that are notexpressed in mice. Without intending to be bound by scientific theory,it is expected that a single IV dose of sFLT1 oligonucleotide willdecrease circulating sFLT1 by 50% within a week and that the effectswill last for 2 weeks or longer. It is expected that decreases incirculating sFLT1 will be associated with modest reduction in bloodpressure. However this will not impair uterine arterial blood flow. Ifdecreases in fetal growth are observed, lowering the dose of sFLT1oligonucleotide therapy may be considered. Alternately, delivering thetherapy as a single dose SQ (which may cause less acute effects on theuterine arterial blood flow) may be considered. If encouraging data isgathered from mouse and initial PK studies in baboons, formalproof-of-concept in vivo efficacy studies will be performed in a baboonmodel of preeclampsia that has been characterized by Dr. AnnemarieHennessy in Australia (Makris, A. et al. Uteroplacental ischemia resultsin proteinuric hypertension and elevated sFLT-1. Kidney international71, 977-984 (2007)). In this study, the different doses and multipledoses of sFlt1 oligonucleotide therapy necessary and sufficient toameliorate preeclampsia signs and symptoms will be specificallyevaluated. Although human sFlt1 ELISA assays cross-react with baboonsFlt1, assays for baboon PlGF and VEGF do not exist. Immunoassays thatreact to baboon PlGF/VEGF are presently being evaluated and, ifsuccessful, free concentrations of PlGF and VEGF levels in blood andurine that may rise as circulating sFlt1 levels decrease will bemeasured as a surrogate of biological efficacy.

6.5 Summary

The compositions and methods described herein will lead to thedevelopment of novel, cost-effective treatment for preeclampsia throughmodulation of sFLT1 levels. In addition, as true for ONTs, technologydeveloped here should be applicable for silencing of other placentalgenes, enabling a wide range of novel functional genomics studies invivo for other pregnancy-related diseases.

Example 6.6. DHA-hsiRNA Conjugates and g2DHA-hsiRNA Conjugates

DHA is an omega-3 fatty acid that is a primary component of the humanbrain (70%) which crosses the blood brain barrier (BBB) and is activelyinternalized by neurons and other cell types. g2DHA (also referred toherein as PC-DHA) is a metabolically active analogue of DHA.

Docosahexaenoic acid (DHA)-hsiRNAs and g2DHA-hsiRNAs (also referred toherein as PC-DHA-hsiRNAs) were synthesized. Tissue distribution ofDHA-hsiRNAs and g2DHA-hsiRNAs post-IV administration (via mouse tailveins) was determined in liver (FIGS. 20 and 21), kidney (FIGS. 22 and23) and placental tissues (FIGS. 24 and 25).

sFLT1 silencing by g2DHA-hsiRNA was observed in pregnant mice using 15mg/kg IV-administered sFLT1_2283P2-g2DHA (150813) in liver, kidney andplacental tissues (FIG. 26).

A particularly preferred sFLT_2283/2519 mix is shown in FIG. 36.

EQUIVALENTS

The disclosure may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the disclosure. Scope of the disclosure is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are therefore intended to be embraced herein.

What is claimed:
 1. A compound comprising an RNA molecule that isbetween 15 and 35 bases in length having a 5′ end, a 3′ end andcomplementarity to an intronic region of an mRNA encoding an sFLT1protein, wherein the RNA molecule comprises a hydrophobic modification.2. The compound of claim 1, comprising double stranded (ds) RNA moleculehaving a sense strand and an antisense strand.
 3. The compound of claim2, wherein the antisense strand comprises a region of complementaritywhich is substantially complementary to (SEQ ID NO: 1)5′ CTCTCGGATCTCCAAATTTA 3′, (SEQ ID NO: 2) 5′ CATCATAGCTACCATTTATT 3′,(SEQ ID NO: 3) 5′ ATTGTACCACACAAAGTAAT 3′ or (SEQ ID NO: 4)5′ GAGCCAAGACAATCATAACA 3′.


4. The dsRNA of claim 3, wherein said region of complementarity iscomplementary to at least 15 contiguous nucleotides of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
 5. The dsRNA of claim 3, whereinsaid dsRNA comprises at least one single stranded nucleotide overhang.6. The dsRNA of claim 5, wherein said dsRNA comprises at least onemodified nucleotide selected from the group consisting of a 2′-O-methylmodified nucleotide, a 2′-fluoro modified nucleotide, a nucleotidecomprising a 5′-phosphorothioate group, a terminal nucleotide linked toa cholesteryl derivative, a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, and a non-natural basenucleotide.
 7. The dsRNA of claim 2, said dsRNA comprising a 5′ end, a3′ end, complementarity to a target, a first oligonucleotide, and asecond oligonucleotide, wherein: (1) the first oligonucleotide comprisesa sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 and SEQ ID NO:4; (2) a portion of the firstoligonucleotide is complementary to a portion of the secondoligonucleotide; (3) the second oligonucleotide comprises alternating2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides; (4) thenucleotides at positions 2 and 14 from the 3′ end of the secondoligonucleotide comprise 2′-methoxy-ribonucleotides; and (5) thenucleotides of the second oligonucleotide are connected viaphosphodiester or phosphorothioate linkages.
 8. The compound of claim 1,wherein the sFLT1 protein is selected from the group consisting of oneor any combination of sFLT1-i13 short, sFLT1-i13 long and sFlt-i15a. 9.A composition comprising a first dsRNA comprising a first sense strandand a first antisense strand and a second dsRNA comprising a secondsense strand and a second antisense strand, wherein the first antisensestrand comprises a first region of complementarity which issubstantially complementary to SEQ ID NO: 1 and the second antisensestrand comprises a second region of complementarity which issubstantially complementary to SEQ ID NO:2.
 10. The composition of claim9, wherein each dsRNA comprises at least one modified nucleotide. 11.The composition of claim 9, wherein each dsRNA comprises a 5′ end, a 3′end and complementarity to a target, wherein: (1) the nucleotidescomprise alternating 2′-methoxy-ribonucleotides and2′-fluoro-ribonucleotides; (2) the nucleotides at positions 2 and 14from the 5′ end are not 2′-methoxy-ribonucleotides; (3) the nucleotidesare connected via phosphodiester or phosphorothioate linkages; and (4)the nucleotides at positions 1-6 from the 3′ end, or positions 1-7 fromthe 3′ end, are connected to adjacent nucleotides via phosphorothioatelinkages.
 12. A pharmaceutical composition comprising: a first dsRNAcomprising a first sense strand and a first antisense strand, whereinthe first antisense strand comprises a region of complementarity whichis substantially complementary to one or both of an intronic region ofsFLT-i13 short and an intronic region of sFLT-i13 long; a second dsRNAcomprising a second sense strand and a second antisense strand, whereinthe second antisense strand comprises a region of complementarity whichis substantially complementary to an intronic region of sFLT-i15a; and apharmaceutically acceptable carrier.
 13. A method of treating ormanaging PE, postpartum PE, eclampsia or HELLP syndrome comprisingadministering to a subject in need of such treatment or management atherapeutically effective amount of the pharmaceutical composition ofclaim
 12. 14. The method of claim 13, wherein the pharmaceuticalcomposition is administered intravenously or subcutaneously.
 15. Amethod of treating one or more symptoms of PE, postpartum PE, eclampsiaor HELLP syndrome in a subject in need thereof, comprising administeringto the subject the composition of claim
 9. 16. A method of treating oneor more symptoms of an angiogenic disorder in a subject in need thereof,comprising administering to the subject the compound of claim
 3. 17. Themethod of claim 16, wherein the angiogenic disorder is selected from thegroup consisting of PE, postpartum PE, eclampsia and HELLP syndrome. 18.The nucleic acid of claim 7, wherein the hydrophobic molecule comprisesa molecule selected from the group consisting of: omega-3 fatty acid,docosanoic acid (DCA), lysophosphatidylcholine esterified DCA (g2-DCA),docosahexaenoic acid (DHA), lysophosphatidylcholine esterified DHA(g2-DHA) and eicosapentaenoic acid (EPA).
 19. The compound of claim 2,wherein the antisense strand comprises a region of complementarity whichcontains no more than 3 mismatches with SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3 or SEQ ID NO:4.
 20. The dsRNA of claim 3, wherein said region ofcomplementarity contains no more than 3 mismatches with SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
 21. The dsRNA of claim 3, whereinsaid region of complementarity is fully complementary to SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
 22. The dsRNA of claim 5,wherein said dsRNA comprises at least one 2′-O-methyl modifiednucleotide, at least one 2′-fluoro modified nucleotide, at least onenucleotide comprising a 5′phosphorothioate group or a terminalnucleotide linked to a cholesteryl derivative.
 23. The dsRNA of claim 7,wherein the second oligonucleotide is linked to the hydrophobic moleculeat the 3′ end of the second oligonucleotide.
 24. The dsRNA of claim 7,wherein the nucleotides at positions 1 and 2 from the 3′ end of secondoligonucleotide are connected to adjacent nucleotides viaphosphorothioate linkages.
 25. The dsRNA of claim 7, the nucleotides atpositions 1 and 2 from the 3′ end of second oligonucleotide, and thenucleotides at positions 1 and 2 from the 5′ end of secondoligonucleotide, are connected to adjacent ribonucleotides viaphosphorothioate linkages.
 26. The pharmaceutical composition of claim12, wherein the first oligonucleotide binds an intronic region of bothsFLT1-i13 short and sFLT1-i13 long.
 27. The pharmaceutical compositionof claim 12, wherein one or both of the first dsRNA or second dsRNAcomprises a hydrophobic modification.
 28. The pharmaceutical compositionof claim 12, wherein the first antisense strand comprises a region ofcomplementarity which is fully complementary to SEQ ID NO:1 and thesecond antisense strand comprises a region of complementarity which isfully complementary to SEQ ID NO:2.